An assay for circulating cell subsets in human peripheral blood by flow cytometry is used as a biomarker to determine cardiovascular disease risk and tumor responsiveness to chemotherapy since endothelial progenitor cells (EPCs) function in vasculogenesis and angiogenesis. Despite analytical advances in polychromatic flow cytometry (PFC), conventional approaches are routinely utilized to enumerate and isolate EPCs, which has led to varied results in clinical studies, potential cellular misidentification, and thus a lack of a plausible biological explanation for how purported EPCs function. Herein, a reproducible PFC protocol is provided to identify a rare circulating endothelial colony‐forming cell (ECFC) with proliferative potential, along with a population of circulating progenitor cells (CPCs) in which the ratio analysis distinguishes between healthy and disease populations. In sum, a reliable PFC protocol, which can be used to investigate the roles of human hematopoietic and endothelial elements in the growth and maintenance of the vasculature, is described. Curr. Protoc. Cytom. 52:9.33.1‐9.33.11. © 2010 by John Wiley & Sons, Inc.