Treg cells, best identified by the expression of the transcription factor FOXP3, play a crucial role in maintaining self-tolerance. Natural Treg cells constitute an independent thymus-derived T-cell lineage whose developmental program in humans is still ill-defined. Here, we provide evidence of a Treg-cell differentiation pathway at the double positive (DP) stage, prior to commitment to the CD4(+) or CD8(+) lineage, in pediatric thymuses. FOXP3(+) DP cells displayed a functional IL-7 receptor and increased Bcl-2 levels that may protect them from cell death/negative selection, and an activated/suppressive phenotype that was lost as CD4 single positive (SP) cells matured and acquired egress markers. A subpopulation of FOXP3(+) DP thymocytes expressing CD103 likely represents the precursor of FOXP3(+) CD8SP cells, which homogeneously expressed this mucosal-homing molecule. Finally, co-cultures of DP thymocytes with primary thymic epithelial cells and multiple linear regression analyses support that FOXP3(+) SP cells are largely derived from FOXP3(+) DP thymocytes. Overall, our data suggest that human Treg-cell lineage commitment significantly occurs at the DP stage with possible implications for the diversity and autoreactivity of the natural Treg-cell repertoire.