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Much progress has been made in the management of chronic-phase chronic myeloid leukemia (CP-CML), but there is a continuing imperative to develop curative treatments, predict patient responses to specific modalities, and anticipate disease relapse or progression. These needs underlie continuing interest in methods to detect and quantify the relevant leukemic cells in clinical samples with improved reliability and specificity. We report the results of comparing three methods to enumerate primitive CP-CML cells in the same samples: genotyping CD34+38− cells directly by fluorescence in situ hybridization, and measuring BCR-ABL1 transcript-genotyped colony-forming cell outputs in either 5-week long-term cultures (LTCs) containing non-engineered mouse fibroblasts or in 6-week LTCs containing mouse fibroblasts engineered to produce human Steel factor, granulocyte colony-stimulating factor, and IL-3. The results demonstrate that the first two methods significantly overestimate the prevalence of primitive CP-CML cells by comparison to the third. In additional studies, we found that CML-CD34+ cells can repopulate the marrow and spleen of serially transplanted adult NOD/SCID-IL-2Rγ chain-null mice for more than 1 year with an almost exclusive myeloid differentiation in primary and secondary recipients and without evidence of disease progression. These findings underscore the importance of long-term functional in vitro and in vivo endpoints to identify and characterize CP-CML stem cells. [Copyright &y& Elsevier]