Regulatory T (T reg) cells play a central role in maintaining immune homeostasis. However, little is known about the stability of T reg cells in vivo. In this study, we demonstrate that a significant percentage of cells exhibited transient or unstable Foxp3 expression. These exFoxp3 + T cells express an activated-memory T cell phenotype, and produced inflammatory cytokines. Moreover, exFoxp3 cell numbers increased in inflamed tissues under autoimmune conditions. Adoptive transfer of autoreactive exFoxp3 cells led to the rapid-onset of diabetes. Finally, T cell receptor repertoire analyses suggested that exFoxp3 cells develop from both natural and adaptive T reg cells. Thus, the generation of potentially autoreactive effector T cells as a consequence of Foxp3 instability has important implications for understanding autoimmune disease pathogenesis.