The study investigates the epigenetic control of the *foxp3* locus in regulatory T cells (Tregs), which play a crucial role in maintaining self-tolerance and preventing autoimmunity. The forkhead transcription factor Foxp3 is essential for Treg development and function, but the molecular mechanisms underlying its stable expression in Tregs are not well understood. The authors identified an evolutionarily conserved region within the *foxp3* locus upstream of exon-1 that exhibits transcriptional activity. Bisulfite sequencing and chromatin immunoprecipitation revealed complete demethylation of CpG motifs and histone modifications in ex vivo isolated Foxp3+CD25+CD4+ Tregs, but not in naive CD25+CD4+ T cells. Partial DNA demethylation was observed in developing Foxp3+ thymocytes, but Tregs induced by TGF-β in vitro displayed only incomplete demethylation despite high Foxp3 expression. These TGF-β-induced Foxp3+ Tregs lost both Foxp3 expression and suppressive activity upon restimulation in the absence of TGF-β. The findings suggest that epigenetic modifications, particularly DNA demethylation and histone modifications, are crucial for stabilizing Foxp3 expression and maintaining the stable suppressor cell lineage of Tregs. This study highlights the importance of epigenetic regulation in the development and function of regulatory T cells and has significant implications for therapeutic applications involving Treg induction or transfer.The study investigates the epigenetic control of the *foxp3* locus in regulatory T cells (Tregs), which play a crucial role in maintaining self-tolerance and preventing autoimmunity. The forkhead transcription factor Foxp3 is essential for Treg development and function, but the molecular mechanisms underlying its stable expression in Tregs are not well understood. The authors identified an evolutionarily conserved region within the *foxp3* locus upstream of exon-1 that exhibits transcriptional activity. Bisulfite sequencing and chromatin immunoprecipitation revealed complete demethylation of CpG motifs and histone modifications in ex vivo isolated Foxp3+CD25+CD4+ Tregs, but not in naive CD25+CD4+ T cells. Partial DNA demethylation was observed in developing Foxp3+ thymocytes, but Tregs induced by TGF-β in vitro displayed only incomplete demethylation despite high Foxp3 expression. These TGF-β-induced Foxp3+ Tregs lost both Foxp3 expression and suppressive activity upon restimulation in the absence of TGF-β. The findings suggest that epigenetic modifications, particularly DNA demethylation and histone modifications, are crucial for stabilizing Foxp3 expression and maintaining the stable suppressor cell lineage of Tregs. This study highlights the importance of epigenetic regulation in the development and function of regulatory T cells and has significant implications for therapeutic applications involving Treg induction or transfer.