The room could be differentiated from the other rooms in the lab due to a rainbow painted in its doorway. Despite its purpose as a place for play, the room itself was originally rather bleak, having plain white walls and hard white flooring.
In later years the room was given a rainbow mural across the walls with several games and activities set out for the children to interact with.
In an effort to retrieve her missing daughter Jane (Eleven), Terry Ives forced her way into Hawkins Lab, threatening the lab's employees with a gun. She eventually made her way to the rainbow room, where she found Jane and Kali playing. However, she was caught by lab personnel before she could take Jane and escape. 9.5 had escaped alongside Three and Six following the security breach.
Eventually, the Rainbow Room was relocated to a more spacious ward of the lab, and painted with rainbow patterns on the wall and floor. The room was also supplied with more tables, toys, and games. During this time, more children were implemented into the program after the three experiments had escaped, including a replacement for the original Six. One day, Brenner had rounded the children into the ward to give them something "very special" planned for them that involved Eleven. The unspecified plan left the rainbow room a bloody mess that was dubbed "The Massacre at Hawkins Lab". One day, according to Kali, Eleven was relocated, and caused Kali to use her powers to escape.
After reuniting with her mother, Eleven was able to see Terry's memory of breaking into the lab and finding the rainbow room. Through this memory, Eleven was able to find and reunite with Kali.
- The room could be an allusion to how rainbows are sometimes connected to MKUltra; some claim the 1939 film The Wizard of Oz was used in the mind control experiments, especially the song “Somewhere Over the Rainbow” featured in the film. However, there is no evidence to support this claim.
- The rainbow room could also be a reference to the 2010 film "Beyond the Black Rainbow", a film that has many similarities to Stranger Things.