Defining the role of epigenetic regulators in hematopoiesis has become critically important, because recurrent mutations or aberrant expression of these genes has been identified in both myeloid and lymphoid hematological malignancies. We found that PRMT4, a type I arginine methyltransferase whose function in normal and malignant hematopoiesis is unknown, is overexpressed in acute myelogenous leukemia patient samples. Overexpression of PRMT4 blocks the myeloid differentiation of human stem/progenitor cells (HSPCs), whereas its knockdown is sufficient to induce myeloid differentiation of HSPCs. We demonstrated that PRMT4 represses the expression of miR-223 in HSPCs via the methylation of RUNX1, which triggers the assembly of a multiprotein repressor complex that includes DPF2. As part of the feedback loop, PRMT4 expression is repressed posttranscriptionally by miR-223. Depletion of PRMT4 results in differentiation of myeloid leukemia cells in vitro and their decreased proliferation in vivo. Thus, targeting PRMT4 holds potential as a novel therapy for acute myelogenous leukemia. Display omitted •PRMT4 blocks myeloid differentiation of human hematopoietic stem/progenitor cells•PRMT4 is downregulated by miR-223 during normal myeloid differentiation•PRMT4 represses miR-223 expression by assembling a methyl-RUNX1-dependent complex•Knockdown of PRMT4 reduces the leukemia cell burden in an AML mouse model Zhao, Nimer, and colleagues now find that the arginine methyltransferase PRMT4 inhibits myeloid differentiation of human stem/progenitor cells. Inhibition occurs through recruitment of a methylation-dependent repressor complex that negatively regulates miR-223 expression. The authors show that PRMT4 is highly expressed in acute myeloid leukemia patient samples and that depletion of PRMT4 reduces leukemia burden in a mouse model. This work provides insights into the role of PRMT4 in normal and malignant hematopoiesis and identifies PRMT4 as an attractive therapeutic target in cancer.