Hematopoietic stem cells form colonies in the spleens of lethally irradiated mice. The number of colonies is indicative of the number of stem cells present in the inoculum. In this article, a technique is presented for effecting a seven-fold enrichment of spleen colony-forming cells. Bone marrow cells are first segregated into “red” and “white” cell populations by centrifugation on Ficoll-Paque. Centrifugation of the “white” cell fraction in 75% Percoll concentrates the colony-forming cells in the top one-third of the gradient. The ability of these cells to repopulate and to cure the anemia of WBB6F1- W/Wv mice indicates that long-term functional pluripotent stem cells have not been destroyed or lost during the fractionation procedures. The segregation procedures enrich the colony-forming cell population from thalassemic as well as from normal mice.