Most AML patients exhibit mutational activation of the PI3K/AKT signaling pathway, which promotes downstream effects including growth, survival, DNA repair, and resistance to chemotherapy. Herein we demonstrate that the inv(16)/KITD816Y AML mouse model exhibits constitutive activation of PI3K/AKT signaling, which was enhanced by chemotherapy-induced DNA damage through DNA-PK-dependent AKT phosphorylation. Strikingly, inhibitors of either PI3K or DNA-PK markedly reduced chemotherapy-induced AKT phosphorylation and signaling leading to increased DNA damage and apoptosis of inv(16)/KITD816Y AML cells in response to chemotherapy. Consistently, combinations of chemotherapy and PI3K or DNA-PK inhibitors synergistically inhibited growth and survival of clonogenic AML cells without substantially inhibiting normal clonogenic bone marrow cells. Moreover, treatment of inv(16)/KITD816Y AML mice with combinations of chemotherapy and PI3K or DNA-PK inhibitors significantly prolonged survival compared to untreated/single-treated mice. Mechanistically, our findings implicate that constitutive activation of PI3K/AKT signaling driven by mutant KIT, and potentially other mutational activators such as FLT3 and RAS, cooperates with chemotherapy-induced DNA-PK-dependent activation of AKT to promote survival, DNA repair, and chemotherapy resistance in AML. Hence, our study provides a rationale to select AML patients exhibiting constitutive PI3K/AKT activation for simultaneous treatment with chemotherapy and inhibitors of DNA-PK and PI3K to improve chemotherapy response and clinical outcome.