The use of flow cytometry has been critical in establishing methods to isolate and characterize hematopoietic stem cells (HSCs) and their progenitors. For more than 30 years, researchers have been uncovering novel markers that when used in combination significantly enhance the purification of HSCs from murine and human bone marrow. The complex interface between HSCs, the lymphohematopoietic system, and their niches, has made identification of HSC markers critical to understanding their biological nature, more so than other adult stem cell populations. Here we review the phenotypic markers and strategies used to purify HSCs, the appropriateness of using these markers for comparisons of HSC function at different stages of ontogeny, and their utility in defining the lineage bias in the HSC compartment.