Previous studies demonstrated that imatinib mesylate (IM) induces autophagy in chronic myeloid leukemia (CML) and that this process is critical to cell survival upon therapy. However, it is not known if the autophagic process differs at basal levels between CML patients and healthy individuals and if pretreatment CML cells harbor unique autophagy characteristics that could predict patients' clinical outcomes. We now demonstrate that several key autophagy genes are differentially expressed in CD34(+) hematopoietic stem/progenitor cells, with the highest transcript levels detected for ATG4B, and that the transcript and protein expression levels of ATG4 family members, ATG5 and BECLIN-1 are significantly increased in CD34(+) cells from chronic-phase CML patients (P < .05). Importantly, ATG4B is differentially expressed in pretreatment CML stem/progenitor cells from subsequent IM responders vs IM nonresponders (P < .05). Knockdown of ATG4B suppresses autophagy, impairs the survival of CML stem/progenitor cells and sensitizes them to IM treatment. Moreover, deregulated expression of ATG4B in CD34(+) CML cells inversely correlates with transcript levels of miR-34a, and ATG4B is shown to be a direct target of miR-34a. This study identifies ATG4B as a potential biomarker for predicting therapeutic response in treatment-naïve CML stem/progenitor cells and uncovers ATG4B as a possible drug target in these cells.