The molecular ordering of the lipids within a biomembrane can profoundly change the local biophysical properties, such as lateral and rotational diffusion, permeability, and fluidity, and these properties, in turn, may affect protein or enzyme activity, transient compartmentalization, and trafficking of vesicular compartments. Specialized nanodomains (or “rafts”) within the plasma membranes of mast cells (immune cells) have been implicated in facilitating the allergic response by compartmentalizing (or excluding) transiently relevant signaling proteins; however they have not yet been observed directly in living cells. We have developed a quantitative optical approach toward the elusive goal of visualizing the spatial and temporal dynamics of these nanodomains with specific signaling molecules. By exploiting the nanoscale sensitivity of fluorescence lifetime and fluorescence resonance energy transfer, we can probe membrane nanodomain dynamics below optical resolution under physiological conditions. We find that the membrane nanostructure changes where antigen-crosslinked high affinity IgE receptor localizes. Importantly these changes correlate well with the functional response, as assessed by stimulated tyrosine phosphorylation of mast cells which initiates the allergic response. Our biophotonics experimental approach should be directly applicable for investigating any signaling pathway that may be dependent upon localize membrane ordering.