Polo-like kinase 1 (Plk1) is a major mitotic regulator overexpressed in many solid tumors. Its role in hematopoietic malignancies is still poorly characterized. In this study, we demonstrate that Plk1 is highly expressed in leukemic cell lines, and overexpressed in a majority of samples from patients with acute myeloid leukemia compared with normal progenitors. A pharmacologic inhibitor, BI2536, blocks proliferation in established cell lines, and dramatically inhibits the clonogenic potential of leukemic cells from patients. Plk1 knockdown by small interfering RNA also blocked proliferation of leukemic cell lines and the clonogenic potential of primary cells from patients. Interestingly, normal primary hematopoietic progenitors are less sensitive to Plk1 inhibition than leukemic cells, whose proliferation is dramatically decreased by the inhibitor. These results highlight Plk1 as a potentially interesting therapeutic target for the treatment of acute myeloid leukemia.