The promiscuous expression of tissue-restricted antigens in the thymus, driven in part by autoimmune regulator (Aire), is critical for the protection of peripheral tissues from autoimmune attack. Aire-dependent processes are thought to promote both clonal deletion and the development of Foxp3⁺ regulatory T (Treg) cells, suggesting that autoimmunity associated with Aire deficiency results from two failed tolerance mechanisms. Here, examination of autoimmune lesions in Aire^−/− mice revealed an unexpected third possibility. We found that the predominant conventional T cell clonotypes infiltrating target lesions express antigen receptors that were preferentially expressed by Foxp3⁺ Treg cells in Aire^+/+ mice. Thus, Aire enforces immune tolerance by ensuring that distinct autoreactive T cell specificities differentiate into the Treg cell lineage; dysregulation of this process results in the diversion of Treg cell-biased clonotypes into pathogenic conventional T cells.