Impaired functional activity of T regulatory cells has been reported in allergic patients and results in an increased susceptibility to autoimmune diseases. The master regulator of T regulatory cell differentiation, the transcription factor FOXP3, is required for both their development and function. Despite its key role, relatively little is known about the molecular mechanisms regulating foxp3 gene expression. In the present study, the effect of Th1 cytokines on human T regulatory cell differentiation was analyzed at epigenetic and gene expression levels and reveals a mechanism by which the STAT1-activating cytokines IL-27 and IFN-gamma amplify TGF-beta-induced FOXP3 expression. This study shows STAT1 binding elements within the proximal part of the human FOXP3 promoter, which we previously hypothesized to function as a key regulatory unit. Direct binding of STAT1 to the FOXP3 promoter following IL-27 stimulation increases its transactivation process and induces permissive histone modifications in this key region of the FOXP3 promoter, suggesting that FOXP3 expression is promoted by IL-27 by two mechanisms. Our data demonstrate a molecular mechanism regulating FOXP3 expression, which is of considerable interest for the development of new drug targets aiming to support anti-inflammatory mechanisms of the immune system.