A novel somatic activating mutation, MPLW515L, has been identified in patients with myelofibrosis with myeloid metaplasia (MF) who are JAK2V617F-negative. This mutation occurs in the transmembrane domain of the thrombopoietin receptor (MPL) and leads to constitutive activation of the JAK-STAT signaling pathway. Expression of MPLW515L in hematopoietic cells results in cytokine-independent growth and thrombopoietin hypersensitivity, and causes constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. A small molecule JAK kinase inhibitor inhibits MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L results in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis, splenomegaly, and increased reticulin fibrosis. These findings suggest that activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD.A novel somatic activating mutation, MPLW515L, has been identified in patients with myelofibrosis with myeloid metaplasia (MF) who are JAK2V617F-negative. This mutation occurs in the transmembrane domain of the thrombopoietin receptor (MPL) and leads to constitutive activation of the JAK-STAT signaling pathway. Expression of MPLW515L in hematopoietic cells results in cytokine-independent growth and thrombopoietin hypersensitivity, and causes constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. A small molecule JAK kinase inhibitor inhibits MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L results in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis, splenomegaly, and increased reticulin fibrosis. These findings suggest that activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD.