The combination of hMSCs with 3D scaffolds has become an important approach to creating functional bone constructs. Bioreactors are important tools to mitigate mass transfer limitations and to provide controlled physiochemical and biomechanical environments for the 3D bone construct development. Media flow in the bioreactor systems is generally controlled either parallel or transverse with respect to the 3D construct, creating different cellular and biomechanical microenvironments in the 3D constructs. In this study, a custom designed modular perfusion bioreactor system was operated under either the parallel or transverse flow. The influence of the flow patterns on the characteristics of the hMSCs' cellular microenvironment and subsequent construct development was investigated. The parallel flow configuration retained ECM proteins and mitogenic growth factors within the scaffold, effectively preserving hMSC progenicity and proliferation potential (e.g., CFU-F, proliferation, and OCT-4), whereas the transverse flow induced hMSC osteogenic differentiation with higher ALP activity and calcium deposition and up-regulation of osteogenic bone markers (e.g., BMP-2, ALP, RUNX2, OSX, and OC). These results demonstrate the regulatory role of the macroscopic flow on the cellular microenvironment of the 3D hMSC construct, and suggest configuring media flow as a strategy for directing hMSC fate and 3D bone construct development in the perfusion bioreactor. Biotechnol. Bioeng. 2012;109: 252–261. © 2011 Wiley Periodicals, Inc.