Aggregation of cell surface receptors is a ubiquitous means of initiating signal transduction in many cellular systems. In this manuscript, we describe a combined theoretical and experimental approach based on multiparameter flow cytometry for measuring the time course of ligand induced aggregation of IgE-FcepsilonRI on RBL cells. By fluorescently labeling both the ligand and surface IgE (sIgE), we have developed an assay that permits us to simultaneously measure both occupancy of sIgE combining sites and association of antigen with the cell surface. This allows for a direct calculation of the degree of receptor aggregation present on the cell. By employing new mixing technologies developed for flow cytometry, we are able to look at aggregation in the sub second time domain. To extend our work, we have synthesized a new set of chemically well defined ligands (of valences 1-3) to use as probes in our studies. We show that the magnitude of the cellular response is dramatically increased as the valence of our ligand is raised from two to three.