Prejudice and discrimination toward gender non-conforming individuals is prevalent and extreme in today’s society. This prejudice can manifest in social exclusion, bullying, and victimization, or physical and sexual assault, and can result in negative social, psychological, academic, and physical health outcomes (e.g., depression, anxiety, suicidality). Thus, it is important to understand the perpetrators of gender expression-based aggression and discrimination. In two studies, I addressed how and why people experience prejudice toward gender non-conforming individuals. Using an affordance management theoretical framework, Study 1 identified threats young adults perceived from gender non-conforming peers. There were differences in perceived threats to personal freedoms, social coordination, and values for gender conforming and non-conforming peers, and these perceptions differed by the political ideology of the perceiver. Study 2 explored children’s threat perceptions associated with gender non-conformity. Children perceived threats to social coordination from gender non-conforming peers but not threats to moral values. Results from both studies supported the use of this theoretical framework for studying prejudice toward gender non-conformity. Together, these studies provide unique information about adults’ and children’s reasons for prejudice toward gender non-conforming peers.