The endosteal niche is critical for the maintenance of hematopoietic stem cells (HSCs). However, it consists of a heterogeneous population in terms of differentiation stage and function. In this study, we characterized endosteal cell populations and examined their ability to maintain HSCs. Bone marrow endosteal cells were subdivided into immature mesenchymal cell-enriched ALCAM⁻Sca-1⁺ cells, osteoblast-enriched ALCAM⁺Sca-1⁻, and ALCAM^–Sca-1⁻ cells. We found that all 3 fractions maintained long-term reconstitution (LTR) activity of HSCs in an in vitro culture. In particular, ALCAM⁺Sca-1⁻ cells significantly enhanced the LTR activity of HSCs by the up-regulation of homing- and cell adhesion–related genes in HSCs. Microarray analysis showed that ALCAM⁻Sca-1⁺ fraction highly expressed cytokine-related genes, whereas the ALCAM⁺Sca-1⁻ fraction expressed multiple cell adhesion molecules, such as cadherins, at a greater level than the other fractions, indicating that the interaction between HSCs and osteoblasts via cell adhesion molecules enhanced the LTR activity of HSCs. Furthermore, we found an osteoblastic marker^low/− subpopulation in ALCAM⁺Sca-1⁻ fraction that expressed cytokines, such as Angpt1 and Thpo, and stem cell marker genes. Altogether, these data suggest that multiple subsets of osteoblasts and mesenchymal progenitor cells constitute the endosteal niche and regulate HSCs in adult bone marrow.