Acute myeloid leukemia (AML) with high ecotropic viral integration site-1 (EVI1) expression (EVI1high AML) is classified as a refractory leukemia with a poor prognosis. We identified G protein-coupled receptor 56 (GPR56) as a novel marker for EVI1high AML, which is an orphan adhesion G protein-coupled receptor (GPCR). GPR56 was found to be associated with high cell adhesion and anti-apoptotic activities in EVI1high AML through activation of RhoA signaling. Moreover, in Gpr56-/- mice, the number of hematopoietic stem cells (HSCs) in bone marrow was significantly decreased with proportional increases in the spleen and peripheral blood, reflecting extramedullary hematopoiesis. The number of Gpr56-/- HSC progenitor cells in the G0 phase was significantly reduced with impaired adhesion and the loss of GPR56 function, which diminished the in vivo repopulating ability of HSCs. In conclusion, GPR56 may represent an important GPCR for the maintenance of quiescence and cellular adhesion of EVI1high AML and HSCs in the bone marrow niche. Moreover, given that GPR56 expression in leukemia stem cells is much higher than that in HSCs, GPR56 is a candidate therapeutic target for leukemia stem cells in EVI1high AML.