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Hematopoiesis is extrinsically controlled by cells of the bone marrow microenvironment, including skeletal lineage cells. The identification and subsequent studies of distinct subpopulations of maturing skeletal cells is currently limited because of a lack of methods to isolate these cells. We found that murine Lin–CD31–Sca-1–CD51+ cells can be divided into 4 subpopulations by using flow cytometry based on their expression of the platelet-derived growth factor receptors ⍺ and β (PDGFR⍺ and PDGFRβ). The use of different skeletal lineage reporters confirmed the skeletal origin of the 4 populations. Multiplex immunohistochemistry studies revealed that all 4 populations were localized near the growth plate and trabecular bone and were rarely found near cortical bone regions or in central bone marrow. Functional studies revealed differences in their abundance, colony-forming unit–fibroblast capacity, and potential to differentiate into mineralized osteoblasts or adipocytes in vitro. Furthermore, the 4 populations had distinct gene expression profiles and differential cell surface expression of leptin receptor (LEPR) and vascular cell adhesion molecule 1 (VCAM-1). Interestingly, we discovered that 1 of these 4 different skeletal populations showed the highest expression of genes involved in the extrinsic regulation of B lymphopoiesis. This cell population varied in abundance between distinct hematopoietically active skeletal sites, and significant differences in the proportions of B-lymphocyte precursors were also observed in these distinct skeletal sites. This cell population also supported pre-B lymphopoiesis in culture. Our method of isolating 4 distinct maturing skeletal populations will help elucidate the roles of distinct skeletal niche cells in regulating hematopoiesis and bone.