In this work a novel method was developed to create a three dimensional environment at a cellular level for bone tissue engineering. Biphasic calcium phosphate (BCP) particles of 140-200 microm were used in association with human mesenchymal stem cells (hMSCs). The cells seeded on these particles adhered and proliferated more rapidly in the first day of culture compared to culture on plastic. Analyses of hMSCs cultured without osteogenic factors on BCP particles revealed an abundant extracellular matrix production forming 3-dimensional (3D) hMSCs/BCP particles constructs after few days. Bone morphogenetic 2 (BMP-2), bone sialoprotein (BSP) and ALP gene expression using real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed that expression profiles were modified by the culture substrate while the addition of osteogenic medium enhanced bone markers expression. These results indicate that BCP particles alone are able to induce an osteoblastic differentiation of hMSCs that might be of interest for bone tissue engineering.