Leukemia stem cells (LSCs) have the capacity to self-renew and propagate disease upon serial transplantation in animal models, and elimination of this cell population is required for curative therapies. Here, we describe a series of pooled, in vivo RNAi screens to identify essential transcription factors (TFs) in a murine model of acute myeloid leukemia (AML) with genetically and phenotypically defined LSCs. These screens reveal the heterodimeric, circadian rhythm TFs Clock and Bmal1 as genes required for the growth of AML cells in vitro and in vivo. Disruption of canonical circadian pathway components produces anti-leukemic effects, including impaired proliferation, enhanced myeloid differentiation, and depletion of LSCs. We find that both normal and malignant hematopoietic cells harbor an intact clock with robust circadian oscillations, and genetic knockout models reveal a leukemia-specific dependence on the pathway. Our findings establish a role for the core circadian clock genes in AML. Display omitted •Normal and malignant hematopoietic cells harbor an intact circadian clock•Circadian rhythm genes Clock and Bmal1 are required for AML cell growth•Disruption of circadian rhythm machinery leads to leukemia stem cell differentiation•Genetic loss of Bmal1 impairs AML maintenance while sparing normal hematopoiesis Disruption of the circadian rhythm machinery in AML produces anti-leukemic effects, including differentiation and depletion of disease-propagating leukemia stem cells.