To understand how the high-affinity IgE receptor (FcΕRI) communicates with downstream effectors, we focused on exploring the functional importance of the FcΕRI-mediated formation and localization of a signaling complex that contains the hematopoietic cell-specific scaffolding protein linker for activation of T cells (LAT) and the guanine nucleotide exchange factor Vav1. Using the mast cell line RBL-2H3, we explored the localization of these proteins by confocal microscopy and cell fractionation. Additionally, the mechanism of function and the importance of LAT and Vav1 to mast cells was studied in genetically disrupted mice and in mast cells derived from their bone marrow. We found that LAT, Vav1 and the adapter molecule SLP-76 associated in detergent-resistant microdomains (lipid rafts) found in the plasma membrane upon FcΕRI stimulation. In the absence of LAT, mast cells showed a remarkable loss of the secretory response and reduced cytokine responses. Vav1 deficiency also affected secretion, although not to the extent of LAT deficiency, and inhibited IL-2 and IFN-γ production. LAT-and Vav1-deficient mice showed reduced blood histamine levels after a systemic anaphylaxis challenge as compared to their normal counterparts. The results demonstrate that LAT is a central mediator in IgE receptor signaling by regulating multiple signaling pathways that affect mast cell degranulation and cytokine production. Vav1, a component of this LAT-containing signaling complex, regulates a specific subset of these responses.