Identification of costimulatory signals required for murine regulatory T (Treg) cell development relies on measuring the frequency of total thymic Treg cells. However, the thymus contains both resident and newly developed Treg cells; whether such signals target both populations is unknown. In this study, we show that CD40-CD154 blockade specifically targeted thymic resident Treg cells, but not, as was previously believed, newly developed Treg cells. Unlike CD28-CD80/CD86 signals, CD40-CD154 signals were not required for Treg cell precursor development. Instead we demonstrate that homeostatic proliferation of thymic resident Treg cells was dependent on CD40-CD154 signals maintaining IL-2 levels. Furthermore, in newborn mice, where all Treg cells are newly developed, blockade of CD40-CD154 signals had no effect on thymic Treg numbers or their proliferation. Our studies highlight the complexity in the study of thymic Treg cell development due to the heterogeneity of thymic Treg cells.