Three cheerleaders join The Troubletones along with Santana and Brittany and dance during the performance of Candyman. They are included in the Christmas Special and performed Christmas Wrapping, dressed as Mother Christmas, with Brittany on lead. Tina and Mike also dances with them. The Cheerios are seen at the football game cheering for the Football Team. Quinn laughs as she watches the team dancing Single Ladies (Put A Ring On It).

When Sue sees Brittany teaching hairography to the other members of the New Directions, with her hair loose, she tells Will that all the Cheerios tie their hair with a high pony because she doesn't want it to the distract them from success.Four cheerleaders dance with Brittany during Dinosaur at prom. It's also in this episode Quinn is out of the wheelchair and is an active cheerleader again. The glee club, convinced by Quinn and Santana, hires Dakota Stanley, a well-known choreographer, to help coach them to Nationals. The cheerleaders hope that the notoriously harsh Stanley will prompt some members of the club to quit, furthering their plan to sabotage the club. Quinn and Santana also lead Mercedes to believe that Kurt has feelings for her, leaving her hurt when he rejects her advances. Sue has the Cheerios help with the Glee Club's Car Wash Fundraiser to help them pay for the new choreographer. Brittany and other cheerleaders dance while Mercedes sings Bust Your Windows. At the end of the episode, Sue is angry that the club is now stronger than ever, and punishes Quinn and Santana. Quinn retaliates by thanking Sue for helping her realize that believing in herself negates the need to bring other people down.

The Cheerios who are also members of The Troubletones performance at Sectionals, singing Survivor/I Will Survive. The club loses and is disbanded. Santana and Brittany rejoin the New Directions.Sue is revealed to be a major Madonna fan, praising her powerful example and asking her Cheerios to emulate Madonna (including dating younger men and using their first names only) so that they will be more empowered in their performance as cheerleaders. The Cheerios perform a routine with stilts to Ray of Light. Kurt and Mercedes join the Cheerios and perform 4 Minutes with them during a school assembly. They tell Will that they were unhappy about never having solos, and will be in both groups. Will reveals that most of the Cheerios are terrible students and should have been failing Spanish for years. Quinn reports to Sue that all of the cheerleaders (except herself, Santana and Brittany) are now academically ineligible after flunking Will's Spanish class. The Cheerios dance during Quinn's performance of You Keep Me Hangin' On. When the other schools perform the songs they had chosen, Quinn reveals that the Cheerios are spying for Sue. Brittany admits that she gave an advance copy of their set-list to Sue, but Santana defends her saying that the Glee Club was the best part of her day. Will tells Principal Figgins this and he suspends Sue as coach.

The Cheerios circle the purple piano, and sprinkle it with lighter fluid her lit cigarette onto the piano, and it bursts into flames. Will tells Santana that because of her sabotage she is banned from New Directions. Their skirts constantly change throughout Season One, from being straight to being pleated for unknown reasons. The Cheerios are seen in the hallway when Jake and Marley see them. Bree has a short and somewhat flirtatious conversation before walking off. They are later seen in a dancing room being coached by Jake. Cheerios members wear their uniform all day long. Some exceptions include Cheerio glee club members performing for some numbers taken place during school hours in the auditorium, in the gymnasium, or in other locations.

After not being eligible to compete in nationals for the Cheerios, Sue claims to have nothing to live for, so Emma suggests that the Cheerios cheer for the teams at their school, which Sue sarcastically responds, "Yeah, like that's gonna happen." However in several episodes such as Preggers and The Sue Sylvester Bowl Shuffle, the Cheerios are seen cheering for the football team.