SLP-76 is an adapter protein expressed in T cells and myeloid cells that is a substrate for ZAP-70 and Syk. SLP-76–deficient mice exhibit a profound block in T-cell development. We found that although SLP-76 is expressed in mouse mast cells, SLP-76–/–mice have normal numbers of mast cells in their skin and bronchi. SLP-76–/–mice are resistant to IgE-mediated passive anaphylaxis. SLP-76–/–mice sensitized with IgE anti-dinitrophenyl (DNP) and then challenged with DNP-HSA developed only mild and transient tachycardia, failed to increase their plasma histamine level, and all survived the antigen challenge. Bone marrow–derived mast cells (BMMCs) from SLP76–/–mice failed to release β-hexosaminidase and to secrete IL-6 after FcεRI cross-linking. Tyrosine phosphorylation of phospholipase C-γ1 (but not of Syk) and calcium mobilization in response to IgE cross-linking were reduced in SLP-76–deficient BMMCs. These results suggest that SLP-76 plays an important role in FcεRI-mediated signaling in mast cells.