T cell receptor (TCR) signaling driven by interaction of the TCR with specific complexes of self-peptide and the major histocompatibility complex determines T cell fate in thymic development. However, the signaling pathway through which TCR signal strength regulates distinct T cell lineages remains unknown. Here we have used mice lacking the endoplasmic reticulum Ca2+ sensors stromal interaction molecule 1 (STIM1) and STIM2 to show that STIM-induced store-operated Ca2+ entry is not essential for thymic development of conventional TCRαβ+ T cells but is specifically required for the development of agonist-selected T cells (regulatory T cells, invariant natural killer T cells, and TCRαβ+ CD8αα+ intestinal intraepithelial lymphocytes). The severe impairment of agonist-selected T cell development is mainly due to a defect in interleukin-2 (IL-2) or IL-15 signaling. Thus, STIM1 and STIM2-mediated store-operated Ca2+ influx, leading to efficient activation of NFAT (nuclear factor of activated T cells), is critical for the postselection maturation of agonist-selected T cells.