TCR-αβ+ double-negative (DN; CD4− CD8−) T cells represent a poorly understood cellular subset suggested to contribute to the pathogenesis of the autoimmune disease systemic lupus erythematosus. DN T cells have been proposed to derive from CD8+ cells. However, the conditions that govern the loss of CD8 expression after Ag encounter are unknown. In this study, we tracked the fate of CD8 T cells from transgenic TCR mice exposed to their cognate Ags as self or in the context of infection. We demonstrate that CD8 T cells lose CD8 expression and become DN only when cognate Ag is sensed as self. This process is restricted to tissues where the Ag is present. We also show that DN T cells derived from self-reactive CD8 cells express the inhibitory molecules PD-1 and Helios. These molecules identify a subset of DN T cells in normal mice. A similar population expands when CD8 T cells from repertoires enriched in self-reactive cells (Aire-deficient) are transferred into cognate hosts. Collectively, our data suggest that a subset of DN T cells, identified by the expression of PD-1 and Helios, represent self-reactive cells. Our results provide an explanation for the origin of DN T cells and introduce CD8 loss as a process associated with self-Ag encounter.