The molecular mechanisms directing the development of 'natural' CD4⁺CD25⁺Foxp3⁺ regulatory T cells (T_reg cells) in the thymus are not thoroughly understood. We show here that conditional deletion of transforming growth factor-β receptor I (TβRI) in T cells blocked the appearance of CD4⁺CD25⁺Foxp3⁺ thymocytes at postnatal days 3–5. Paradoxically, however, beginning 1 week after birth, the same TβRI-mutant mice showed accelerated expansion of thymic CD4⁺CD25⁺Foxp3⁺ populations. This rapid recovery of Foxp3⁺ thymocytes was attributable mainly to overproduction of and heightened responsiveness to interleukin 2, as genetic ablation of interleukin 2 in TβRI-mutant mice resulted in a complete absence of CD4⁺CD25⁺Foxp3⁺ cells from the thymus and periphery. Thus, transforming growth factor-β signaling is critical to the thymic development of natural CD4⁺CD25⁺Foxp3⁺ T_reg cells.