An analytical study of cell-cell communications between murine osteoblast-like MLO-A5 cells and bone marrow mesenchymal stem cell (BMSC)-like C3H10T1/2 cells was performed. C3H10T1/2 cells expressing green fluorescent protein (10T-GFP cells) were generated to enable the isolation of the BMSC-like cells from co-cultures with MLO-A5 cells. The mRNA expression levels of several osteogenic transcription factors (Runx2, Osterix, Dlx5, and Msx2) did not differ between the co-cultured and mono-cultured 10T-GFP cells, but those of alkaline phosphatase (ALP) and bone sialoprotein (BSP) were 300- to 400-fold higher in the co-cultured cells. Patch clamp and biocytin transfer assays revealed gap junction-mediated communication between co-cultured 10T-GFP and MLO-A5 cells. The addition of a gap junction inhibitor suppressed the increases in the expression levels of the ALP and BSP mRNAs in co-cultured 10T-GFP cells. Furthermore, the histone acetylation levels were higher in co-cultured 10T-GFP cells than in mono-cultured 10T-GFP cells. These results suggest that osteoblasts and BMSCs associate via gap junctions, and that gap junction-mediated signaling induces histone acetylation that leads to elevated transcription of the genes encoding ALP and BSP in BMSCs.