CD4⁺CD25⁺ regulatory T (T reg) cells play a pivotal role in control of the immune response. Transforming growth factor-β (TGF-β) has been shown to be required for T reg cell activity; however, precisely how it is involved in the mechanism of suppression is poorly understood. Using the T cell transfer model of colitis, we show here that CD4⁺CD45RB^high T cells that express a dominant negative TGF-β receptor type II (dnTβRII) and therefore cannot respond to TGF-β, escape control by T reg cells in vivo. CD4⁺CD25⁺ T reg cells from the thymus of dnTβRII mice retain the ability to inhibit colitis, suggesting that T cell responsiveness to TGF-β is not required for the development or peripheral function of thymic-derived T reg cells. In contrast, T reg cell activity among the peripheral dnTβRII CD4⁺CD25⁺ population is masked by the presence of colitogenic effector cells that cannot be suppressed. Finally, we show that CD4⁺CD25⁺ T reg cells develop normally in the absence of TGF-β1 and retain the ability to suppress colitis in vivo. Importantly, the function of TGF-β1^−/− T reg cells was abrogated by anti–TGF-β monoclonal antibody, indicating that functional TGF-β can be provided by a non–T reg cell source.