In bone tissues, gap junctions form direct links between the cytoplasm of an osteocyte and another adjacent osteocyte or osteoblast, which underlie both bone formation and bone resorption. We have previously demonstrated that alkaline phosphatase (ALP) and bone sialoprotein (BSP), which are osteoblast markers, were induced in mesenchymal stem cells (MSCs) co-cultured with osteoblast-like cell line. However, the molecular mechanism of this process has not been fully addressed. Furthermore, few advances have been made toward elucidating the communication networks that link the status of committed cells such as (pre-) adipocytes that differentiated from MSCs as well as osteoblasts. Therefore, the objective of the present study was to investigate the mechanism underlying the communication network between pre-adipocytes and osteoblasts. We evaluated the effect of co-culture with osteoblast on the cell status of pre-adipocytes using murine osteoblast-like cell line, MLO-A5, and pre-adipocyte-like cell line, 3T3-L1, respectively. The results presented here demonstrated that osteoblasts and pre-adipocytes communicate via gap junctions, and the ensuing drastic increase in ALP and BSP transcription in co-cultured pre-adipocytes was induced, at least partly, via heat shock protein family B member 1 (Hspb1). In addition, terminal differentiation into adipocytes was suppressed in pre-adipocytes during co-culture with osteoblast without loss of adipogenic differentiation ability. Interestingly, after co-culture with osteoblasts, isolated co-cultured pre-adipocytes were able to differentiate to adipocytes as well as original pre-adipocytes. These results suggest that gap junctional communication with osteoblasts suppressed adipogenic differentiation of pre-adipocytes without loss of adipogenic differentiation ability.