Background & Aims: Regulatory T (Treg) cells are plastic, but the in vivo mechanisms by which they are converted into foxhead box p3 (Foxp3 +) interferon (IFN)-γ+ T cells and whether these converted cells retain the ability to inhibit colitis are not clear. Methods: Foxp3+ Treg cells were generated by culture of nave CD4+ T cells from Foxp3GFP CBir1 T-cell receptor (TCR) transgenic (Tg) (CBir1-Tg) mice, which are specific for CBir1 flagellin (an immunodominant microbiota antigen), with transforming growth factor-β. Foxp3 GFP+ CBir1-Tg Treg cells were isolated by fluorescence-activated cell sorting and transferred into TCRβxδ-/- mice. Colitis was induced by transfer of nave CBir1-Tg CD4+ T cells into immunodeficient mice. Results: Microbiota antigen-specific Foxp3+ Treg cells were converted, in the intestine, to IFN-γ+ T-helper (Th)1 cells, interleukin (IL)-17+ Th17 cells, and Foxp3+ T cells that coexpress IFN-γ and/or IL-17. Conversion of Treg cells into IFN-γ-producing Th1 cells and Foxp3+IFN-γ+ T cells required innate cell production of IL-12 in the intestine; blocking IL-12 with an antibody inhibited their conversion to Th1 and Foxp3+IFN- γ+ T cells in the intestines of mice that were recipients of Treg cells. Addition of IL-12, but not IL-23, promoted conversion of Treg cells into Th1 and Foxp3+IFN-γ+ T cells, in vitro. Foxp3+IFN-γ+ T cells had regulatory activity because they suppressed proliferation of nave T cells, in vitro, and inhibited induction of colitis by microbiota antigen-specific T cells. IFN-γ+ Th1 cells were not converted into Treg cells; Foxp3 +IFN-γ+ T cells differentiated into IFN-γ+ but not Foxp3+ T cells. Conclusions: IL-12 promotes conversion of Treg cells into IFN-γ-expressing cells; Foxp3 +IFN-γ+ T cells retain their regulatory functions and develop during the transition of Foxp3+ Treg cells into IFN-γ+ Th1 cells. © 2011 AGA Institute.