Regulatory T cell (Treg) stability has been shown to be maintained by the epigenetic modifications, such as DNA demethylation of Treg-specific demethylation regions (TSDRs) during Treg development. Recently, it has been reported that DNA demethylase, ten-eleven translocation (TET) family proteins are involved in the DNA demethylation of TSDRs, and thus regulating stability of Tregs. However, the role of TET proteins in the peripheral differentiation of Tregs as well as other T helper subsets has not been elucidated. To investigate the role of TET proteins in T cell differentiation, we generated T cell-specific TET2 and TET3 double-knockout (Tet2f/fTet3f/fCd4-Cre; DKO) mice. DKO mice died at an average of 10 weeks of age with splenomegaly and lymph adenopathy accompanied by uncontrolled activation of T cells and B cells. Most T cells in DKO mice were activated and exhibited Th17- and/or follicular helper T (Tfh)-type phenotypes, while Treg population was very small in these mice. T cell expansion was dependent on gut microbiota, suggesting that DKO T cells recognized antigens of intestinal bacteria. We also found that the majority of Tregs in colon lamina propria (cLP) in DKO mice express Helios, a marker for thymic-derived Tregs (tTregs), suggesting that TET2/3-deficiency in CD4+ T cells failed to differentiate into peripheral Tregs (pTregs). This was further confirmed in DKO mice with Rag2-/-OTII-Tg background; there was a high population of Th17 cells but only a few pTregs after feeding with OVA. Furthermore, naive T cells isolated from DKO mice differentiated into less iTregs but more Th17 cells compared to those from wild type mice in vitro. These data indicate that TET2 and TET3 regulate Treg/Th17 differentiation in periphery. Molecular mechanism is under investigation.