Tyrosine kinase inhibitors (TKIs) only partially inhibit the growth of Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (Ph B-ALL) cells, and often lead to rapid relapse. Therefore, it is essential to elucidate the mechanisms of resistance and develop novel treatment strategies. Histone deacetylases (HDACs) are often dysregulated in hematological malignancies, and many HDAC inhibitors have shown potent antitumor activities. In this study, we found that HDAC8 was highly expressed in Ph B-ALL patient samples upon TKI treatment. HDAC8 inhibition significantly increased TKI-mediated elimination of leukemia cells and decreased their ability to initiate leukemia. Using two mouse models, we demonstrated that TKIs in combination with targeting HDAC8 effectively inhibited leukemia progression and reduced the frequencies of stem cells. Mechanistically, HDAC8 deacetylated hypoxia inducible factor-1α (HIF-1α) at lysine 19/21, leading to a reduction in PHD2-mediated hydroxylation and subsequent pVHL-mediated ubiquitination, which slowed the degradation of HIF-1α. HIF-1α inhibition induced apoptosis and decreased the initiating capacity of leukemia cells. Importantly, in a Ph B-ALL patient-derived xenograft model, targeting HDAC8 or HIF-1α in combination with TKIs significantly inhibited leukemia progression. In conclusion, our study revealed that targeting HDAC8/HIF-1α in combination with TKIs could be a promising strategy for treating Ph B-ALL.