Foxp3 is the master transcriptional regulator of regulatory T cells (Tregs), and the stabilization of Foxp3 expression is regulated by the demethylation of conserved non-coding sequence 2 (CNS2) in the Foxp3 locus. Recent studies have shown that TCR stimulation is required for the demethylation of Foxp3 CNS2 during Treg development. However, the relationship between the strength of TCR stimulation and the demethylation of Foxp3 CNS2 remains unclear. To address this issue, we compared the frequency of demethylation of the Foxp3 CNS2 among in vitro-induced Tregs (iTreg) that had received a range of TCR stimulation during their development. We found that the frequency of demethylation of the Foxp3 CNS2 was increased with increased TCR stimulation strength, whereas CD28 stimulation had only a limited effect. Mechanistically, the binding of Tet2, a member of the TET family of enzymes involved in DNA demethylation, on the Foxp3 CNS2 was increased by strong TCR stimulation. Furthermore, compared with iTreg induced by weak TCR stimulation, iTreg induced by strong TCR stimulation maintained Foxp3 expression both in vitro and in vivo. These data indicate that the strength of TCR stimulation is a key factor for induction of the demethylation of Foxp3 CNS2 and the generation of stable Tregs. (C) 2018 Elsevier Inc. All rights reserved.