The MST1 kinase was recently identified as playing an essential role in the promotion of lymphocyte polarization and adhesion stimulated by chemokines and TCR signaling. However, the physiological relevance of the Mst1 pathway in thymocyte development is not completely understood. In this study, we analyzed the effect of Mst1 disruption on thymocyte development and migration. Mst1-deficient (Mst1−/−) mice displayed an accumulation of mature thymocytes in the thymus, a dramatic reduction of lymphocytes in blood and peripheral lymphoid tissues, and a decrease of homing ability to peripheral lymph nodes. Mst1−/− thymocytes were impaired in chemotactic response to chemokines, such as CCL19, but not to sphingosine-1-phosphate. Further analyses of Mst1−/− mice revealed a severe impairment in the egress of mature T cells from the thymus. T lineage-specific knockout of the Mst1 gene demonstrates a cell-intrinsic role for Mst1 in regulating T cell development. Our study indicates that Mst1 is crucial in controlling lymphocyte chemotaxis and thymocyte emigration.