Characterization of defects in a variant subline of RBL mast cells has revealed a biochemical event proximal to IgE receptor (FcεRI)-stimulated tyrosine phosphorylation that is required for multiple functional responses. This cell line, designated B6A4C1, is deficient in both FcεRI-mediated degranulation and biosynthesis of several lipid raft components. Agents that bypass receptor-mediated Ca²⁺ influx stimulate strong degranulation responses in these variant cells. Cross-linking of IgE-FcεRI on these cells stimulates robust tyrosine phosphorylation but fails to mobilize a sustained Ca²⁺ response. FcεRI-mediated inositol phosphate production is not detectable in these cells, and failure of adenosine receptors to mobilize Ca²⁺ suggests a general deficiency in stimulated phospholipase C activity. Antigen stimulation of phospholipases A₂ and D is also defective. Infection of B6A4C1 cells with vaccinia virus constructs expressing constitutively active Rho family members Cdc42 and Rac restores antigen-stimulated degranulation, and active Cdc42 (but not active Rac) restores ganglioside and GPI expression. The results support the hypothesis that activation of Cdc42 and/or Rac is critical for FcεRI-mediated signaling that leads to Ca²⁺ mobilization and degranulation. Furthermore, they suggest that Cdc42 plays an important role in the biosynthesis and expression of certain components of lipid rafts.