Elias Toufexis was originally cast only as Leonidas and not as Nikolaos. He stood in for Nikolaos in motion capture and Ubisoft had the intention of replacing his voice later with another actor. When Ubisoft saw his performance they decided to have him fully performance capture both parts.
This is the first Assassin's Creed game in which player choices would have a severe impact on history.
The development team originally planned to make Kassandra the only playable character until they were told (by the marketing department or an editorial creative lead) it wasn't an option.
In the beginning of the game during the Battle of Thermopylae, King Leonidas can be seen throwing his shield at an enemy. While this game is by no means supposed to be historically accurate, this is a glaring error because Spartans would never give up their shield, they would rather die on it than lose it.
Sam Fisher's iconic night vision goggles from Ubisoft's Splinter Cell video game franchise can be found in Layla Hassan's safehouse. Hassan also briefly comments on it saying that it belongs to the Fourth Echelon.