The study identifies a novel somatic activating mutation, MPLW515L, in the thrombopoietin receptor (MPL) gene in a subset of patients with myelofibrosis with myeloid metaplasia (MF) who are JAK2V617F-negative. MPLW515L mutation is found in 9% of JAK2V617F-negative MF patients and is not detected in JAK2V617F-negative essential thrombocythemia (ET) or polycythemia vera (PV) patients. The mutation results in cytokine-independent growth and constitutive activation of the JAK-STAT signaling pathway in hematopoietic cells. In a murine bone marrow transplant model, expression of MPLW515L induces a myeloproliferative disorder characterized by thrombocytosis, leukocytosis, splenomegaly, and increased reticulin fibrosis. A small molecule JAK kinase inhibitor inhibits MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. These findings suggest that MPLW515L is a pathogenetic mutation in JAK2V617F-negative MF and that targeted inhibition of JAK-STAT signaling may be an effective therapeutic strategy for this subset of patients.The study identifies a novel somatic activating mutation, MPLW515L, in the thrombopoietin receptor (MPL) gene in a subset of patients with myelofibrosis with myeloid metaplasia (MF) who are JAK2V617F-negative. MPLW515L mutation is found in 9% of JAK2V617F-negative MF patients and is not detected in JAK2V617F-negative essential thrombocythemia (ET) or polycythemia vera (PV) patients. The mutation results in cytokine-independent growth and constitutive activation of the JAK-STAT signaling pathway in hematopoietic cells. In a murine bone marrow transplant model, expression of MPLW515L induces a myeloproliferative disorder characterized by thrombocytosis, leukocytosis, splenomegaly, and increased reticulin fibrosis. A small molecule JAK kinase inhibitor inhibits MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. These findings suggest that MPLW515L is a pathogenetic mutation in JAK2V617F-negative MF and that targeted inhibition of JAK-STAT signaling may be an effective therapeutic strategy for this subset of patients.