The mammalian Hippo signaling pathway has been implicated in the self-renewal and differentiation of stem and progenitor cells. MST1 and MST2 (MST1/2) are core serine-threonine kinases in the Hippo signaling pathway, one of which, MST1, has been extensively investigated for its role in T cell and myeloid cell function. These studies have identified MST1 as a promising therapeutic target in immunological disease. However, the roles of MST1/2 in hematopoietic stem cell (HSC) function in vivo are not fully understood. Here, we report that mice with a conditional deletion of Mst1/2 exhibit impaired hematopoietic stem and progenitor cell (HSPC) function under stress condition. Furthermore, Mst1/2 deletion markedly altered mature cell output. Therefore, MST1/2 are indispensable for maintenance as well as function of stem and progenitor cells under steady state conditions and with transplantation stress.