The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement of T cells. LAT is also expressed in platelets, NK, and mast cells. Although LAT-deficient mice contain normal numbers of mast cells, we found that LAT-deficient mice were resistant to IgE-mediated passive systemic anaphylaxis. LAT-deficient bone marrow–derived mast cells (BMMC) showed normal growth and development. Whereas tyrosine phosphorylation of FcεRI, Syk, and Vav was intact in LAT-deficient BMMCs following FcεRI engagement, tyrosine phosphorylation of SLP-76, PLC-γ1, and PLC-γ2 and calcium mobilization were dramatically reduced. LAT-deficient BMMCs also exhibited profound defects in activation of MAPK, degranulation, and cytokine production after FcεRI cross-linking. These results show that LAT plays a critical role in FcεRI-mediated signaling in mast cells.