The infiltration of adipocytes in osteoporotic patients' bone marrow suggests an important regulatory function of bone marrow fat on the development of aged bone. Therefore, we have examined the effects of adipocytes derived from bone mesenchymal stem cell (MSC) on osteoblast differentiation using two different co-culture modes (direct mode and indirect mode). Alkaline phosphatase (ALP)-positive areas and mineralized areas of MSC-derived osteoblasts decrease similarly in the two co-culture modes as the amount of MSC-derived adipocytes increases, suggesting that the crosstalk between adipocytes and osteoblasts may be mainly through secretory factors in the medium. To further understand the molecular mechanisms, both mRNA and protein expressions in osteoblasts in the lower layer of the indirect mode were analyzed, leading to identification of 12 differential genes/proteins. Among them, S100A6 and calreticulin are possibly related to bone formation. S100A6 was down-regulated and calreticulin was up-regulated as MSC-derived adipocytes increased. Similarly, differential expression of these proteins was also observed in bone tissue slides from young (1-month-old) and old (6-month-old) mice. The expression level of β-catenin in osteoblasts of bone tissues was lower in 6-month-old mice compared to 1-month-old mice. Total TGF-β analyzed with antibody-based protein microarray and active TGF-β analyzed with ELISA in the co-cultured cell medium increased consistently as the amount of adipocytes increased. Taken together, our results suggest that MSC-derived adipocytes may regulate osteoblast differentiation in the aged bone through TGF-β-mediated canonical Wnt signaling.