MicroRNA-9 (miR-9) is emerging as a critical regulator of organ development and neurogenesis. It is also deregulated in several types of solid tumors; however, its role in hematopoiesis and leukemogenesis is not yet known. Here we show that miR-9 is detected in hematopoietic stem cells and hematopoietic progenitor cells, and that its expression increases during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis and promotes apoptosis in vitro and in vivo. Conversely, in hematopoietic progenitor cells, the inhibition of miR-9 with a miRNA sponge blocks myelopoiesis. Ecotropic viral integration site 1 (EVI1), required for normal embryogenesis, is considered an oncogene because its inappropriate up-regulation induces malignant transformation in solid and hematopoietic cancers. Here we show that EVI1 binds to the promoter of miR-9-3, leading to DNA hypermethylation of the promoter and repression of miR-9. Moreover, miR-9 expression reverses a myeloid differentiation block that is induced by EVI1. Our findings indicate that EVI1, when inappropriately expressed, delays or blocks myeloid differentiation at least in part by DNA hypermethylation and down-regulation of miR-9. It was reported that Forkhead box class O genes (FoxOs) inhibit myeloid differentiation and prevent differentiation of leukemia-initiating cells. Here we identify both FoxO1 and FoxO3 as direct targets of miR-9 in hematopoietic cells and find that up-regulation of FoxO3 inhibits miR-9-induced myelopoiesis. These results reveal a unique role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms and provide insights into the epigenetic regulation of miR9 in tumorigenesis.