Mesenchymal stem cells (MSCs) are multipotent stem cells predominantly obtained from bone marrow, which are sensitive to mechanical loadings in physiological microenvironment. However, how the MSCs sense and respond to extremely low fluidic shear stress analogous to interstitial flow in vivo is poorly understood. In this work, we present a functional microfluidic device to examine the migration and differentiation behaviors of MSCs in response to multiple orders of physiologically relevant interstitial flow levels. The different magnitudes of fluid flow-induced shear stress were produced by a hydraulic resistance-based microfluidic perfusion system consisting of a microchannel network and a parallel of uniform cell culture chambers. By changing the length and width of the flow-in channels, the multiple magnitudes of low shear stresses could be generated ranging from ~10 to ~10 dyne/cm . We demonstrated enhanced significant F-actin expression and cell migration in MSCs under applied fluidic shear stress at ~10 dyne/cm . We also demonstrated a significant osteogenic differentiation under this interstitial level of slow flows from ~10 to ~10 dyne/cm in MSCs by analyzing alkaline phosphatase activity and osteopontin staining. Moreover, cytochalasin D and Rho-inhibitor Y-27632 significantly reduced the cytoskeleton F-actin expression and osteogenic differentiation in MSCs, indicating the mediated mechanical responses of MSCs under extremely low fluidic shear stress, possibly as a consequence of Rho-associated kinase pathway. The established microfluidic perfusion system with multiple shear-flow capabilities is simple and easy to operate, providing a flexible platform for studying the responses of diverse types of cells to the multiple interstitial flow levels in a single assay.