Tyrosine kinase inhibitor treatment of chronic myeloid leukemia (CML) has demonstrated beneficial effects. However, resistance to tyrosine kinase inhibitors and disease relapse are still a challenge for CML therapy. In this study, we analyzed bone marrow samples from 149 CML patients and 15 control donors, and investigated the affect of AF1q on CML cell survival and engraftment in vitro and in vivo. We found that AF1q/MLLT11 expression was significantly upregulated in CML patients, especially in CD34+ CML cells. Elevated AF1q expression was associated with disease progression. Knockdown of AF1q enhanced imatinib sensitivity, induced apoptosis, and suppressed growth in CML cells. Moreover, AF1q deficiency sensitized CD34+ CML cells to imatinib. In contrast, upregulation of AF1q promoted cell survival, protected CML cells from imatinib-induced apoptosis, and increased engraftment of CML cells in vivo. We further identified a positive correlation between AF1q and CD44 expression in chronic phase CML patients and CD34+ CML cells. Importantly, AF1q contributes to imatinib-resistance in CML by regulating the expression of CD44. These findings reveal a novel BCR-ABL-independent pathway, AF1q/CD44, involves imatinib resistance in CML, thus representing a potential therapeutic target for imatinib-resistant CML patients.