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Inside the Assassin Tomb beneath Santa Maria della Visitazione, if Ezio stares into the water near the first lever for about a minute, a cutscene plays showing a giant squid swimming past. Look again and a tentacle nearly slaps him.
When Ezio first meets his uncle Mario Auditore, Mario greets him with "It's-a-me! Mario!", a direct lift of the first line spoken by Mario in Super Mario 64.
While free-running or climbing, a male bystander occasionally mutters "Another capering crusader," a nod to Batman, who is nicknamed the Caped Crusader and shares Ezio's love of rooftops and stealth.
After meeting Niccolò Machiavelli, returning to the Palazzo Auditore courtyard and using Eagle Vision triggers a one-time-only vision of Ezio's entire deceased family standing around the palazzo.
Looting practice dummies during the missions "Knowledge Is Power" and "Hunting the Hunter" grants the player keys, leftover, dummied-out content that serves absolutely no purpose in the game.
Throw coins into a crowd and female bystanders will sometimes shout "Money! That's what I want!", a reference to the Motown classic of the same name.
If every guard in the Doge's Palace is pre-assassinated before confronting Carlo Grimaldi, he flees to the bottom of the Giants' Staircase and cowers, lip-syncing dialogue lifted straight from the game's original E3 demo.
After assassinating Carlo Grimaldi, heading to the Venice dock area lets players find Da Vinci's flying machine still lying wrecked where it crashed earlier in the story.
Glitching into the Palazzo Ducale's private quarters reveals the exact chess position from the cutscene where Carlo Grimaldi assassinated Doge Giovanni Mocenigo, though the candle they played under is oddly unlit, and the sky outside turns blood red, likely an unintended lighting bug.
Subject 16 hid twenty encoded Glyphs across the Animus recreation of Renaissance Italy, five in Florence, one in Monteriggioni, five in Tuscany, two in Romagna, and seven in Venice, each unlocking a fragment of the video known as "The Truth."
