Objective: Osteoporosis ,characterized by damaged bone metabolism, poses a significant challenge to public healthcare. A promising therapeutic approach involves bolstering osteogenic differentiation and mitigating osteoporosis through the transplantation of bone marrow mesenchymal stem cells (BMSCs). In this study, we aimed to elucidate BMSC heterogeneity and the underlying mechanisms of BMSC differentiation.Methods: ScRNA-seq profiles of endosteal bone marrow samples from one-, three- and sixteen-month-old male Col2/Td mice were curated from the GEO. Quality control and cell annotation were performed by Scater and Seurat packages. CellChat was employed for inferring intercellular communication. Pseudo-time analysis was presented using Monocle tool. BMSC differentiation-related genes were screened with Mfuzz package. GSVA was used for functional enrichment analysis. Results: Within the BMSCs, we identified the presence of adipocytes, chondrocytes, myeloid progenitor cells, osteoblasts, endothelial cells, smooth muscle cells. Among these, adipocytes and chondrocytes emerged as central cell types, based on ligand-receptor interaction strength.Pseudo-time analysis unveiled age-dependent alterations in chondrocyte differentiation,and we identified genes linked to chondrocyte differentiation, with a pronounced enrichment in metabolic pathways.Conclusion: Our findings identified BMSC subpopulations and uncovered the metabolic alterations during chondrocyte differentiation.