Human bone marrow stromal cells (hMSCs) are a promising source of multipotent progenitors for a broad range of stem cell therapies for regenerative medicine. A basic prerequisite to realizing the therapeutic potential of hMSCs is the ability to identify stem-like progenitors in a heterogeneous culture. To overcome current limitations in stem cell identification, we have developed a novel high-capacity assay that will characterize the heterogeneity inherent to hMSCs by (1) classifying cells according to colony-forming efficiency as a measure of proliferation capacity and trilineage potential as a measure of multipotency and (2) preserving a frozen template of these cell populations for future use. Specifically, the assay quantifies the percentage of tripotent progenitors, bipotent progenitors, lineage-committed unipotent progenitors and mature cells in an hMSC culture. The high-capacity format facilitates the analysis of large sample sizes required to obtain statistically significant data on hMSC populations. The assay has numerous clinical and basic research applications, for example, to monitor hMSC preparations in the clinic for consistent content of stem-like progenitors and to evaluate the effect of culture conditions on progenitor content in the laboratory. This work was funded with grants from NSF and NIH.