Antigen-mediated cross-linking of the high affinity receptor for IgE (FcεRI), in the plasma membrane of mast cells, is the first step in the allergic immune response. This event triggers the phosphorylation of specific tyrosines in the cytoplasmic segments of the β and γ subunits of FcεRI by the Src tyrosine kinase Lyn, which is anchored to the inner leaflet of the plasma membrane. Lyn-induced phosphorylation of FcεRI occurs in a cholesterol-dependent manner, leading to the hypothesis that cholesterol-rich domains, or "lipid rafts," may act as functional platforms for IgE receptor signaling. Testing this hypothesis under physiological conditions remains challenging because of the notion that these functional domains are likely transient and much smaller than the diffraction limit of optical microscopy. Here we use ultrafast fluorescence dynamics to investigate the correlation between nanostructural changes in the plasma membrane (labeled with 1,1′-dioctadecyl-3,3,3′,3′- tetramethylindocarbocyanine (diI-C18)) and IgE-FcεRI cross-linking in adherent RBL mast cells stimulated with multivalent antigen. Time-dependent two-photon fluorescence lifetime imaging microscopy of diI-C 18 shows changes in lifetime that agree with the kinetics of stimulated tyrosine phosphorylation of FcεRI, the first identifiable biochemical step of the allergic response, under the same conditions. In addition, two-photon fluorescence lifetime imaging microscopy of Alexa Fluor 488-labeled IgE indicates that Förster resonance energy transfer occurs with diI-C18 in the plasma membrane. Our live cell studies provide direct evidence for the association of IgE-FcεRI with specialized cholesterol-rich domains within ∼4-nm proximity and with an energy transfer efficiency of 0.22±0.01 at maximal association during IgE receptor signaling.