In mast cells, cross-linking the high-affinity IgE receptor (FcεRI) initiates the Lyn-mediated phosphorylation of receptor ITAMs, forming phospho-ITAM binding sites for Syk. Previous immunogold labeling of membrane sheets showed that resting FcεRI colocalize loosely with Lyn, whereas cross-linked FcεRI redistribute into specialized domains (osmiophilic patches) that exclude Lyn, accumulate Syk, and are often bordered by coated pits. Here, the distribution of FcεRI β is mapped relative to linker for activation of T cells (LAT), Grb2-binding protein 2 (Gab2), two PLCγ isoforms, and the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), all implicated in the remodeling of membrane inositol phospholipids. Before activation, PLCγ1 and Gab2 are not strongly membrane associated, LAT occurs in small membrane clusters separate from receptor, and PLCγ2, that coprecipitates with LAT, occurs in clusters and along cytoskeletal cables. After activation, PLCγ2, Gab2, and a portion of p85 colocalize with FcεRI β in osmiophilic patches. LAT clusters enlarge within 30 s of receptor activation, forming elongated complexes that can intersect osmiophilic patches without mixing. PLCγ1 and another portion of p85 associate preferentially with activated LAT. Supporting multiple distributions of PI3-kinase, FcεRI cross-linking increases PI3-kinase activity in anti-LAT, anti-FcεRIβ, and anti-Gab2 immune complexes. We propose that activated mast cells propagate signals from primary domains organized around FcεRIβ and from secondary domains, including one organized around LAT.