It has been shown that TGF-ss1 is critical in the generation of CD4+CD25+Foxp3+ inducible regulatory T cells (iTregs) from naܯve CD4+T cells. However, in contrast to natural Tregs, TGF-ss1-induced iTregs rapidly lose both Foxp3 expression and suppression activity. We found that TGF-ss1-induced Foxp3 levels were maintained by the addition of the anti-IL-4 antibody or by STAT6 gene deletion. Thus, IL-4 is an important suppressor of Foxp3 induction, and Th2 development is a major cause for the disappearance of iTreg during long culture. Using promoter analysis in EL4 cells and primary T cells, we identified a silencer region containing a STAT6 binding site. STAT6 binding to this site reduced TGF-ss1-mediated Foxp3 promoter activation and chromatin modification. Retinoic acid (RA) has also been shown to suppress loss of Foxp3 induced by TGF-ss1. RA in the presence of TGF-ss1 reduced STAT6 binding to the Foxp3 promoter and enhanced histone acetylation, thereby reverting the effect of IL-4. We propose that antagonistic agents for neutralizing IL-4 could be a novel strategy to facilitate inducible Treg cell generation and the promotion of tolerance in Th2-dominated diseases such as allergy.