Jak2 tyrosine kinase is essential for animal development and hyperkinetic Jak2 function has been linked to a host of human diseases. Control of this pathway using Jak2-specific inhibitors would therefore potentially serve as a useful research tool and/or therapeutic agent. Here, we used a high-throughput program called DOCK to predict the ability of 20,000 small molecules to interact with a structural pocket adjacent to the ATP-binding site of murine Jak2. One small molecule, 2-methyl-1-phenyl-4-pyridin-2-yl-2-(2-pyridin-2-ylethyl)butan-1-one (herein designated as Z3), bound to Jak2 with a favorable energy score. Z3 inhibited Jak2-V617F and Jak2-WT autophosphorylation in a dose-dependent manner but was not cytotoxic to cells at concentrations that inhibited kinase activity. Z3 selectively inhibited Jak2 kinase function with no effect on Tyk2 or c-Src kinase function. Z3 significantly inhibited proliferation of the Jak2-V617F-expressing, human erythroleukemia cell line, HEL 92.1.7. The Z3-mediated reduction in cell proliferation correlated with reduced Jak2 and STAT3 tyrosine phosphorylation levels as well as marked cell cycle arrest. Finally, Z3 inhibited the growth of hematopoietic progenitor cells isolated from the bone marrow of an essential thrombocythemia patient harboring the Jak2-V617F mutation and a polycythemia vera patient carrying a Jak2-F537I mutation. Collectively, the data suggest that Z3 is a novel specific inhibitor of Jak2 tyrosine kinase.