The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1-deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte-associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function. Author summary T cells are pivotal in orchestrating an adaptive immune response. They are produced in the thymus and undergo selection processes resulting in elimination of nonfunctional and self-reactive cells in order to prevent autoimmune disease. One type of T cells, called regulatory T cells (Treg cells), is generated either in the thymus or in the periphery through a process termed agonist selection. Peripheral Treg cells are crucial for the suppression of unwanted immune responses. However, too much suppressive function of Treg cells can also impair immune responses directed against tumors. Here, we have analyzed the mechanisms that regulate this process and show that a microRNA, miR-181a/b-1, controls de novo generation of Treg cells by modulating agonist selection. We show that thymic and peripheral Treg cells deficient in miR-181a/b-1 contain higher levels of the key effector molecule CTLA-4, and as a consequence, such Treg cells display increased suppressive capacity. We observe that the expression of miR-181a/b-1 in peripheral Treg cells is much lower when compared to thymocytes;thus, our study implies that peripheral Treg-cell effector function is imprinted during intrathymic differentiation.