Transcription factor Foxp3 (forkhead box P3), restricted in its expression to a specialized regulatory CD4+ T-cell subset (T R) with a dedicated suppressor function, controls T R lineage development. In humans and mice, Foxp3 deficiency results in a paucity of T R cells and a fatal breach in immunological tolerance, causing highly aggressive multi-organ autoimmune pathology 1, 2, 3. Here, through genome-wide analysis combining chromatin immunoprecipitation with mouse genome tiling array profiling, we identify Foxp3 binding regions for∼ 700 genes and for an intergenically encoded microRNA. We find that a large number of Foxp3-bound genes are up-or downregulated in Foxp3+ T cells, suggesting that Foxp3 acts as both a transcriptional activator and repressor. Foxp3-mediated regulation unique to the thymus affects, among others, genes encoding nuclear factors that control gene expression and chromatin remodelling. In contrast, Foxp3 target genes shared by the thymic and peripheral T R cells encode primarily plasma membrane proteins, as well as cell signalling proteins. Together, our studies suggest that distinct transcriptional sub-programmes implemented by Foxp3 establish T R lineage during differentiation and its proliferative and functional competence in the periphery.