The application of flow cytometry for the study of cell surface antigens has evolved significantly in the last 20 yr. One of the factors involved is the continuing development of a vast array of monoclonal antibodies specific for cell-associated antigens. Another important factor is the development of newer fluorochromes, such as phycoerythrin (PE) and peridinin chlorophyll (PERCp), or tandem conjugates, such as PE-Cy5, that complement fluorescein isothiocyanate (FITC) to allow simultaneous measurement of coexpression of three different antigens. With the availability of dyes that allow for three- and now four-color analyses, along with the demand from investigators for this ability, the major manufacturers of flow cytometry instrumentation have produced instruments that readily measure three fluorescence colors along with forward and side scatter. Simple bench-top flow cytometers capable of four-color measurements have made their way to the marketplace.