Mast cells have key functions as effectors of immunoglobulin E–mediated allergic inflammatory diseases. Allergen stimulation induces Ca²⁺ influx and elicits the secretion of inflammatory mediators from mast cells. Here we show that the Ca²⁺-binding endoplasmic reticulum protein STIM1 is critical to mast cell function. STIM1-deficient fetal liver–derived mast cells had impaired Ca²⁺ influx mediated by the high-affinity immunoglobulin E receptor FcεRI and activation of the transcription factors NF-κB and NFAT. Mast cells lacking STIM1 also had much less degranulation and cytokine production after FcεRI stimulation. In addition, alterations in STIM1 expression affected the sensitivity of immunoglobulin E–mediated immediate-phase anaphylactic responses in vivo. Thus, STIM1 is key in promoting the Ca²⁺ influx that is essential for FcεRI-mediated mast cell activation and anaphylaxis.