Store-operated Ca²⁺ entry through calcium release–activated calcium channels is the chief mechanism for increasing intracellular Ca²⁺ in immune cells. Here we show that mouse T cells and fibroblasts lacking the calcium sensor STIM1 had severely impaired store-operated Ca²⁺ influx, whereas deficiency in the calcium sensor STIM2 had a smaller effect. However, T cells lacking either STIM1 or STIM2 had much less cytokine production and nuclear translocation of the transcription factor NFAT. T cell–specific ablation of both STIM1 and STIM2 resulted in a notable lymphoproliferative phenotype and a selective decrease in regulatory T cell numbers. We conclude that both STIM1 and STIM2 promote store-operated Ca²⁺ entry into T cells and fibroblasts and that STIM proteins are required for the development and function of regulatory T cells.