NK cells from the blood of chronic myelogenous leukemia (CML) patients are progressively decreased in number as the disease progresses from chronic phase to blast crisis. We hypothesize that BCR/ABL may be directly responsible by interfering with NK cell differentiation. CD34＋HLA-DR＋cells from CML patients were studied for their capacity to differentiate into NK cells. The NK cell cloning frequency was significantly decreased from CML CD34＋HLA-DR＋cells compared with cells from normal donors, yet CD34＋HLA-DR＋cells gave rise to BCR/ABL NK cells in some patients. This finding prompted us to further investigate circulating NK cells from the blood of CML patients. CD56＋CD3- NK cells were sorted from CML patients and examined by fluorescence in situ hybridization (FISH). In contrast to chronic phase CML, significant numbers of NK cells from advanced phase CML patients were BCR/ABL, whereas T cells were always BCR/ABL-regardless of the disease stage. To test the effects of BCR/ABL as the sole genetic abnormality, BCR/ABL was transduced into umbilical cord blood CD34 cells, and NK development was studied. p210-enhanced green fluorescence protein-transduced cells gave rise to significantly decreased numbers of NK cells compared with enhanced green fluorescence protein transduction alone. In addition, the extrinsic addition of BCR/ABL-transduced autologous CD34＋cells suppressed the NK cell differentiation of normal umbilical cord blood CD34＋CD38- cells. This study provides the first evidence that BCR/ABL is responsible for the altered differentiation of NK cells and that the NK cell lineage can be involved with the malignant clone in advanced stage CML. The Journal of Immunology, 2002, 168: 643-650.