Endomucin (EMCN) currently represents the only hematopoietic stem cell (HSC) marker expressed by both murine and human HSCs. Here, we report that EMCN⁺ long-term repopulating HSCs (LT-HSCs; CD150⁺CD48⁻LSK) have a higher long-term multi-lineage repopulating capacity compared to EMCN⁻ LT-HSCs. Cell cycle analyses and transcriptional profiling demonstrated that EMCN⁺ LT-HSCs were more quiescent compared to EMCN⁻ LT-HSCs. Emcn^−/− and Emcn^+/+ mice displayed comparable steady-state hematopoiesis, as well as frequencies, transcriptional programs, and long-term multi-lineage repopulating capacity of their LT-HSCs. Complementary functional analyses further revealed increased cell cycle entry upon treatment with 5-fluorouracil and reduced granulocyte colony-stimulating factor (GCSF) mobilization of Emcn^−/− LT-HSCs, demonstrating that EMCN expression by LT-HSCs associates with quiescence in response to hematopoietic stress and is indispensable for effective LT-HSC mobilization. Transplantation of wild-type bone marrow cells into Emcn^−/− or Emcn^+/+ recipients demonstrated that EMCN is essential for endothelial cell-dependent maintenance/self-renewal of the LT-HSC pool and sustained blood cell production post-transplant.