The mammalian Hippo signaling pathway has been implicated in oncogenesis in the context of solid tumors such as hepatocellular carcinoma. Mammalian sterile 20–like kinase 1 (MST1), the core component of the Hippo signaling pathway, is highly expressed in hematopoietic cells. However, its possible impact on tumorigenesis in this setting is unknown. In this study, we provide evidence that Mst1 loss in the mouse enhances chemically and genetically induced lymphoma development by inducing chromosomal instability. Mst1 deficiency increased susceptibility to T-cell acute lymphoblastic leukemia induced by mutagen exposure. Notably, before transformation Mst1^−/− normal thymocytes showed no changes in proliferation or apoptosis in vitro and in vivo, but they displayed elevated levels of abnormal mitotic chromosomes and aneuploidy, conditions known to promote tumorigenesis. Mst1^−/− mice also showed accelerated formation of spontaneous lymphomas in a p53-deficient background, accompanied by severe aneuploidy. In clinical specimens of lymphoma and leukemia, we documented frequent downregulation of MST1 expression, consistent with our findings. Taken together, our findings reveal a tumor suppressive function of Mst1 based on its ability to prevent chromosomal instability in lymphocytes. Cancer Res; 72(20); 5386–95. ©2012 AACR.