Immunological tolerance to self requires naturally occurring regulatory T (T-reg) cells. Yet how they stably control autoimmune T cells remains obscure. Here, we show that T-reg cells can render self-reactive human CD8(+) T cells anergic (i.e., hypoproliferative and cytokine hypoproducing upon antigen restimulation) in vitro, likely by controlling the costimulatory function of antigen-presenting cells. Anergic T cells were naive in phenotype, lower than activated T cells in T cell receptor affinity for cognate antigen, and expressed several coinhibitory molecules, including cytotoxic T lymphocyte-associated antigen-4 (CTLA-4). Using these criteria, we detected in healthy individuals anergic T cells reactive with a skin antigen targeted in the autoimmune disease vitiligo. Collectively, our results suggest that T-reg cell-mediated induction of anergy in autoimmune T cells is important for maintaining self-tolerance.