With Lucy Stillman's failure to obtain the Apple of Eden for the Templars, Warren Vidic initialized the Animi Training Program, a project to train Abstergo Industries' agents with the skills necessary to combat the Assassins.
Contained within the program were the genetic record of influential Templars and individuals, also referred to as Animi Avatars, with there being 21 available for use (in total) in the first stage. A few of the Avatars' counterparts were also present as targets for Ezio Auditore to kill during the Renaissance in Rome, as a part of Templar agent side memories.
- Barber - Wearing ordinary noble's clothes and displaying white streaks in his dark hair, the Barber used a razor to assassinate his targets quickly and efficiently.
- Blacksmith - A large man wearing armor and sporting a noticeable, bright red beard, the Blacksmith used a large hammer as his weapon, allowing him to crush his target's heads with the same force of how he struck metal.
- Captain - Wearing decorative armor and possessing refined sword skills, the Captain wielded a thin rapier and killed his targets with stylish flair.
- Courtesan - A sensual woman in liberal clothing, the Courtesan charmed her targets before assassinating them in fluid, but lethal movements, with her special bladed fan.
- Dama Rossa (DLC exclusive) - A young, but serious looking leather clad woman, the Dama Rossa killed her targets similarly to a black widow spider, by using her double pronged hair pin.
- Doctor - Bearing dark robes and a beak-shaped mask to hide his face, the Doctor used a syringe to assassinate his targets and patients, killing many more than what he saved.
- Engineer - An expert marksman, wearing mostly red and white clothes with puffy sleeves, the Engineer wielded a drafting compass as his weapon, and killed his targets with careful preparation.
- Executioner - A muscular looking man with the appearance of a butcher, the Executioner wielded a handheld axe as his weapon, allowing him to kill his targets with a brutal display.
- Footpad - A man dressed in the garb of a bandit, the Footpad sank his pair of shivs into the flesh of his target, in a manner reminiscent of a snake, before disappearing from the scene of the crime.
- Harlequin (Pre-order and DLC exclusive) - Wearing a costume similar to those of the performers of Carnevale, the Harlequin adorned himself with a mask to hide his identity and carried two thin, needle-like rondel daggers, allowing him to make a show out of his target's murder.
- Hellequin (Uplay reward) - Wearing a purple and yellow striped costume, which resembled the outfit of her brother, the Harlequin, the Hellequin donned an intricately detailed mask to partially cover her face, and used her two barbed daggers to deliver her target a dramatic end.
- Knight (DLC exclusive) - Covered in head to toe in strong brass armor, the Knight used his titanic greatsword to forcefully execute his targets, taking down the Templar's enemies easily and obediently.
- Marquis (DLC exclusive) - An affluent man with links to royalty, the Marquis used his sabre to take down his targets with ease, as if they were nothing but a simple distraction.
- Mercenary - A strong man who bore a heavy resemblance to Mario Auditore and other condottiero, the Mercenary wielded a short sword and used it to kill his targets, showing no mercy as he drove its wide blade deep into their bodies.
- Nobleman - Wearing fine clothing and a gold-lined cape, the Nobleman assassinated his targets using his claw, a prosthetic construct set in place of his missing arm.
- Officer (Pre-order and DLC exclusive) - A sleek, middle-aged man, adorned in a fine black trenchcoat, the Officer wielded an epieu and used it to silence his targets, warding off any oppression to the Templars' cause.
- Pariah (DLC exclusive) - A wealthy, but stocky looking man – wearing a metal mask and body-covering robes – the Pariah killed his targets with no hesitation, by pulling the trigger on his gunpowder-activated bolt gun.
- Priest - A bald man wearing robes similar to those of a monk, the Priest used a very thin holy blade as his weapon, in order to deliver his target's salvation.
- Prowler - Using skills and wearing robes very similar to the Assassins, though sporting Templar affiliations, the Prowler used a retracting switchblade to kill his targets, being a Templar prototype of the Hidden Blade.
- Smuggler - A woman who wore a hooded robe and trafficked rare items, the Smuggler possessed an inverted Hidden Blade, which held some similarity to the weapons of the Assassins and allowed her to kill her targets without them making a sound.
- Thief - A woman wearing ragged clothes, the Thief wielded the sharp half of a pair of scissors in a makeshift scissor blade, using it on her targets to exact vengeance for her brother's death.
- There were three civilian types in the first stage of the Animi Training Program, being heralds, merchants, and performers, and each were displayed in multiple variations.
- Every Animi Avatar, aside from the Thief and the four Da Vinci Disappearance Avatars, featured in the first chapter of the Rome pack in Assassin's Creed: Project Legacy.
- When killed in the single player, Templar agents entered the Memory Corridor, but they did not converse with Ezio, unlike the main assassination targets.
- As well as this, the Templar agents did not use the weapons that their Animi Avatar counterparts used in the Animi Training Program.
- Despite having their own unique moves, Animi Avatars could all break their target's necks in the same fashion as one another.