We have previously reported that primitive normal hematopoietic cells detectable as long-term culture-initiating cells (Ph-LTC-IC) are present at high levels in the blood of some patients with chronic myeloid leukemia (CML). We now show that this population can be expanded several-fold when highly purified CD34⁺CD38⁻ cells isolated from the blood of such patients are cultured for 10 days in a serum-free medium containing 100 ng/mL of Flt3-ligand and Steel factor and 20 ng/mL of interleukin-3 (IL-3) and IL-6, and granulocyte colony-stimulating factor. In similar cultures initiated with CD34⁺CD38⁻ cells from CML blood samples in which all of the LTC-IC were leukemic (Ph⁺), Ph⁺ LTC-IC activity was rapidly lost both in the presence and absence of admixed CD34⁺CD38⁻ cells isolated from normal marrow. Conversely, the ability of normal LTC-IC to expand their numbers was shown to be independent of the presence of Ph⁺LTC-IC and later types of Ph⁺colony-forming cell (CFC) progenitors. In contrast to the LTC-IC, CFC were consistently -a m p l i f i e d  i n  c u l t u r e s  i n i t i a t e d  w i t h  C M L - d e r i v e d -CD34⁺CD38⁻ cells and the additional CFC present after 10 days were, like the starting population of CFC, almost exclusively Ph⁺ regardless of the genotype(s) of the LTC-IC in the original CML samples. Amplification of the Ph⁺CFC population in these cultures showed the same factor dependence as previously demonstrated for the in vitro expansion of CFC from normal marrow CD34⁺CD38⁻ cells. Ph⁺LTC-IC disappeared regardless of the cytokines present. Taken together these findings support a model of CML in which the leukemic stem cells are characterized by a decreased probability of self-renewal and an increased probability of differentiation. In addition, they suggest new opportunities for improving the treatment of CML using strategies that require autologous stem cell rescue.