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Compelling evidence suggests that the transcription factor Foxp3 acts as a master switch governing the development and function of CD4þ regulatory T cells (Tregs). However, whether transcriptional control of Foxp3 expression itself contributes to the development of a stable Treg lineage has thus far not been investigated. We here identified an evolutionarily conserved region within the foxp3 locus upstream of exon-1 possessing transcriptional activity. Bisulphite sequencing and chromatin immunoprecipitation revealed complete demethylation of CpG motifs as well as histone modifications within the conserved region in ex vivo isolated Foxp3þCD25þCD4þ Tregs, but not in naı̈ve CD25CD4þ T cells. Partial DNA demethylation is already found within developing Foxp3þ thymocytes; however, Tregs induced by TGF-b in vitro display only incomplete demethylation despite high Foxp3 expression. In contrast to natural Tregs, these TGF-b–induced Foxp3þ Tregs lose both Foxp3 expression and suppressive activity upon restimulation in the absence of TGF-b. Our data suggest that expression of Foxp3 must be stabilized by epigenetic modification to allow the development of a permanent suppressor cell lineage, a finding of significant importance for therapeutic applications involving induction or transfer of Tregs and for the understanding of long-term cell lineage decisions