Acute myeloid leukemia (AML) remains a devastating disease in need of new therapies to improve patient survival. Targeted, adoptive T cell therapies have achieved impressive clinical outcomes in some B-cell leukemias and lymphomas but not in AML. Double negative T cells (DNTs) effectively kill blast cells from the majority of AML patients and are now being tested in clinical trials. However, AML blasts obtained from ~30% of patients show resistance to DNT cell-mediated cytotoxicity; the markers or mechanisms underlying this resistance have not been elucidated. Here, we used a targeted CRISPR/Cas9 screen to identify genes that confer susceptibility of AML cells to DNT cell therapy. Inactivation of the SAGA deubiquitinating complex components sensitized AML cells to DNT-mediated cytotoxicity. In contrast, CD64 inactivation resulted in resistance to DNT-mediated cytotoxicity. Importantly, the level of CD64 expression strongly correlated with the sensitivity of AML cells to DNT cell treatment. Furthermore, the ectopic expression of CD64 overcame AML resistance to DNTs both in vitro and in vivo. Altogether, our data demonstrate the utility of CRISPR/Cas9 screens to uncover mechanisms underlying the sensitivity to DNT cell therapy and suggest CD64 as a predictive marker for response in AML patients.