The present study combines chemical and mechanical stimuli to modulate the osteogenic differentiation of mesenchymal stem cells (MSCs). Arg–Gly–Asp (RGD) peptides incorporated into biomaterials have been shown to upregulate MSC osteoblastic differentiation. However, these effects have been assessed under static culture conditions, while it has been reported that flow perfusion also has an enhancing effect on MSC osteoblastic differentiation. It is clear that there is a need to combine RGD modification of biomaterials with mechanical stimulation of MSCs via flow perfusion and evaluate its effects on MSC differentiation down the osteogenic lineage. In this study, the effect of different levels of RGD modification of poly(L‐lactic acid) scaffolds on MSC osteogenesis was evaluated under conditions of flow perfusion. It was found that there is a synergistic enhancement of different osteogenic markers, due to the combination of flow perfusion and RGD surface modification when compared to their individual effects. Furthermore, under conditions of flow perfusion, there is an RGD surface concentration optimal for differentiation, and it is flow rate‐dependent. This report underlines the significance of incorporating combined biomimesis via biochemical and mechanical microenvironments that modulate in vivo cell behaviour and tissue function for more efficient tissue‐engineering strategies. Copyright © 2010 John Wiley & Sons, Ltd.