Little is known about the ontogeny of naturally occurring CD4⁺CD25⁺ regulatory/suppressor T cells that play a major role in maintaining self-tolerance in mice and humans. In rodents, thymectomy on day 3 of life leads to multiple organ-specific autoimmune diseases that can be prevented by adoptive transfer of regulatory T cells, suggesting their neonatal development. We investigated regulatory T-cell ontogeny in 11 human fetuses. Together with the first mature T cells, thymic CD4⁺CD25⁺ cells were detected as early as 13 weeks of gestation. Thymic CD25⁺ cells appeared to be positively selected at the CD4⁺CD8⁺CD3^hi differentiation stage, as assessed by CD1a and CD69 expression. The proportion of thymic CD4⁺CD25⁺ cells appeared quite stable with age, around 6% to 7%, similar to the proportion observed in infant thymi. Extrathymic CD4⁺CD25⁺ T cells could hardly be detected at 13 weeks of gestation but were present from week 14 onwards. As adult regulatory T cells, purified CD4⁺CD25⁺ fetal cells were anergic and suppressed T-cell proliferative responses; they expressed intracellular cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and Foxp3 mRNA. Altogether, our results indicate that the generation of regulatory/suppressor T cells is consubstantial to the generation of a functional and self-tolerant immune system. (Blood. 2005;105:4715-4721)