CD4⁺Foxp3⁺ regulatory T cells (T_reg cells) are largely autoreactive yet escape clonal deletion in the thymus. We demonstrate here that CD27–CD70 co-stimulation in the thymus rescues developing T_reg cells from apoptosis and thereby promotes T_reg cell generation. Genetic ablation of CD27 or its ligand CD70 reduced T_reg cell numbers in the thymus and peripheral lymphoid organs, whereas it did not alter conventional CD4⁺Foxp3⁻ T cell numbers. The CD27–CD70 pathway was not required for pre-T_reg cell generation, Foxp3 induction, or mature T_reg cell function. Rather, CD27 signaling enhanced positive selection of T_reg cells within the thymus in a cell-intrinsic manner. CD27 signals promoted the survival of thymic T_reg cells by inhibiting the mitochondrial apoptosis pathway. CD70 was expressed on Aire⁻ and Aire⁺ medullary thymic epithelial cells (mTECs) and on dendritic cells (DCs) in the thymic medulla. CD70 on both mTECs and DCs contributed to T_reg cell development as shown in BM chimera experiments with CD70-deficient mice. In vitro experiments indicated that CD70 on the CD8α⁺ subset of thymic DCs promoted T_reg cell development. Our data suggest that mTECs and DCs form dedicated niches in the thymic medulla, in which CD27–CD70 co-stimulation rescues developing T_reg cells from apoptosis, subsequent to Foxp3 induction by TCR and CD28 signals.