Ecotropic viral integration site 1 (Evi1) is a transcription factor that is highly expressed in hematopoietic stem cells and is crucial for their self-renewal capacity. Aberrant expression of Evi1 is observed in 5% to 10% of de novo acute myeloid leukemia (AML) patients and predicts poor prognosis, reflecting multiple leukemogenic properties of Evi1. Here, we show that thrombopoietin (THPO) signaling is implicated in growth and survival of Evi1-expressing cells using a mouse model of Evi1 leukemia. We first identified that the expression of megakaryocytic surface molecules such as ITGA2B (CD41) and the THPO receptor, MPL, positively correlates with EVI1 expression in AML patients. In agreement with this finding, a subpopulation of bone marrow and spleen cells derived from Evi1 leukemia mice expressed both CD41 and Mpl. CD41(+) Evi1 leukemia cells induced secondary leukemia more efficiently than CD41(-) cells in a serial bone marrow transplantation assay. Importantly, the CD41(+) cells predominantly expressing Mpl effectively proliferated and survived on OP9 stromal cells in the presence of THPO via upregulating BCL-xL expression, suggesting an essential role of the THPO/MPL/BCL-xL cascade in enhancing the progression of Evi1 leukemia. These observations provide a novel aspect of the diverse functions of Evi1 in leukemogenesis.