Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-FcepsilonRI and Lyn-EGFP on individual cells. We also simultaneously measure temporal variations in the lateral diffusion of these proteins. Antigen-stimulated interactions between these proteins detected subsequent to the initiation of receptor phosphorylation exhibit time-dependent changes, suggesting multiple associations between FcepsilonRI and Lyn-EGFP. During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity. These stimulated interactions are not observed between FcepsilonRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI implicated in downstream signaling events. They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein-protein interactions in intact, living cells.