0Ⅹ, the third and last book in Piers Anthony's Of Man and Manta series, features a reference to the Game of Life in the form of a race of Energy Beings operating according to cellular automata rules.
Glory Season by David Brin features this game with the variation of being played as a contest, with each side getting part of the board to use, with the goal being eliminating the other side.
In Riddle of the Seven Realms, a character creates a dimension that operates under these rules, even calling it "the realm of the conways."
The WWW Trilogy's first book has Caitlin seeing cellular automata in the background of her "websight;" Dr. Kuroda brings up the Game of Life as an example of cellular automata while explaining it.
One of the intro cinematics for Darwinia is a simulation of Life, with the added caveats that the grid is confined to a finite space and each Darwinian will die after a set number of years no matter what. As it's incredibly difficult not to get attached to the little guys while playing Darwinia, seeing the last "block" and "spaceship" formations settle, flicker, and die can be very haunting. In-universe, it was given to the Darwinians to teach them mortality.
Kingdom of Loathing has a mushroom farming minigame that follows the same rules (although with more than one kind of mushroom), but the board is only 4x4, making it impossible to create any particularly interesting patterns.
Entering the cheat "gol" in SimCity 4 plays the Game of Life using SimCity 4's grid-based lot system.
In System Shock, the walls, floors and ceilings of the virtual Cyber Space environment are square grids that light up in patterns that are seemingly arbitrary, until you realize they're following rules from the Game of Life.
ZUN: You might not understand this if you didn't study at a technical college.
The Powder Toy has a particle that follows Game of Life rules, along with a few other similar particles that follow slight modifications of the rules, such as HLIF (High Life) which replicates when 3 or 6 cells are alive next to a space, rather than just 3.
Yume 2kki has a room in Data Stream that references the game.
In "Strip Games", the author Google searches "Strip Conway's Game of Life". Unsurprisingly, there were no results.
"RIP John Conway" is an animation in which a cell pattern resembling a human stick figure develops into a single glider, which travels off the edge of the grid and disappears, as a memorial to the recently-deceased Conway.
The Orion's Arm Project has, among artifacts of unclear origin, a planet sized Life field. Apparently, it's been played long enough for life to evolve in the game.
makes reference to the bullet patterns being inspired by those found in Conway's Game of Life.ZUN: You might not understand this if you didn't study at a technical college.