The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and is associated with poor prognosis. In such patients, FLT3 tyrosine kinase inhibitors (TKIs) are only partially effective and do not eliminate the leukemia stem cells (LSCs) that are assumed to be the source of treatment failure. Here, we show that the NAD-dependent SIRT1 deacetylase is selectively overexpressed in primary human FLT3-ITD AML LSCs. This SIRT1 overexpression is related to enhanced expression of the USP22 deubiquitinase induced by c-MYC, leading to reduced SIRT1 ubiquitination and enhanced stability. Inhibition of SIRT1 expression or activity reduced the growth of FLT3-ITD AML LSCs and significantly enhanced TKI-mediated killing of the cells. Therefore, these results identify a c-MYC-related network that enhances SIRT1 protein expression in human FLT3-ITD AML LSCs and contributes to their maintenance. Inhibition of this oncogenic network could be an attractive approach for targeting FLT3-ITD AML LSCs to improve treatment outcomes. Display omitted •SIRT1 protein expression is selectively enhanced in FLT3-ITD AML stem cells•Combined SIRT1 and FLT3 inhibition increases elimination of FLT3-ITD AML stem cells•SIRT1 overexpression in FLT3-ITD AML is related to c-MYC activation•c-MYC increases SIRT1 expression by enhancing expression of the USP22 deubiquitinase Li et al. find that c-MYC via USP22 deubiquitinase enhances SIRT1 expression and promotes maintenance of human AML stem cells, which suggests a strategy to target leukemia stem cells.