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 FcepsilonRI and activation of the transcription factors NF-kappaB and NFAT. Mast cells lacking STIM1 also had much less degranulation and cytokine production after FcepsilonRI 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 FcepsilonRI-mediated mast cell activation and anaphylaxis.