According to the Marine campaign, the game takes place in the year "2145 — ten years after the Hadley's Hope incident", referring to the events seen in Aliens. However, 2145 is actually 34 years before the events of Aliens, which takes place in 2179.
Although blurry and indistinct, the USS Tyrargo appears to have the name "SULACO" written on its hull.
The M56 Smartgun's tracking mechanisms are infrared in nature. However, in Aliens, the Xenomorphs are shown to be invisible to infrared scanning (similarly, Yautja are unable to detect them with their heat-based vision mode). The Smartgun should therefore be unable to track the creatures, yet in the game the weapon is able to lock on to and track the creatures with ease.
During the Marine campaign, Corporal Harrison encounters Prince in a stasis pod within the Implantation Lab and unintentionally releases him. Later, Harrison sees Prince jump onto a dropship and escape. Before witnessing this event, Harrison appears to leave the pod where he discovered Prince and enter another. However, in the Predator campaign, Prince jumps onto the dropship from the same pod where he is released.
Similarly to Aliens versus Predator, the M56 Smartgun in the game is able to track Xenomorphs, despite the fact they should be invisible to its infrared scanning.
In the Gear Room, the Disc's description reads "This throwing weapon homes in on its target with lethal precision, it's spinning blades cutting our prey in half." The word "it's" should be spelt "its", as it is earlier in the sentence.
In the Predator campaign, when the Predator protagonist passes a window, he stops and the game's Alien protagonist arrives and jumps on the glass before leaving. However, in the Alien campaign, the game's Predator protagonist runs by the glass without stopping and the Alien instead stops at the window, without jumping on it.
The sign outside Hadley's Hope gives the colony's population as 159. However, in Aliens, the colony's population is 158 (given both on the sign outside in the extended Special Edition, and when Ripley tells Burke he is responsible for 157 deaths — 157 + Newt = 158).
Towards the end of issue 1, Davis One states that, in addition to himself, there are six Davis units still operational aboard the Europa following the battles with the Xenomorphs. However, by studying the identifiable head numbers of the Davis units in the next two issues, at least eight different Davis units appear — 02, 03, 04 (also identified in dialogue), 07, 09, 18, 21 and 27.
While Chestburster implantation has often been portrayed inconsistently across the franchise, taking anywhere between 24 hours and ten minutes, Defiance #6 seemingly shows it happening literally in moments. Specifically, Hollis is attacked by a horde of Facehuggers while Zula is attempting to open a door so that she can escape; the door is open in mere seconds, yet in that time a Facehugger has seemingly attacked Hollis, as she is seen pushing the parasite from her face with slime/blood around her lips. She is alter shown to have been impregnated with a Chestburster, despite the fleeting contact.
When attempting to view a page that doesn't exist, the message "SPECIAL ORDER 404 PRIORITY ONE INSURE RETURN OF WEBPAGE FOR ANALYSIS. ALL OTHER CONSIDERATIONS SECONDARY. CONTENT EXPENDABLE." is displayed. Although this was intentional as a reference to Special Order 937 in Alien, "INSURE" should be "ENSURE".