Physical loading is a potent stimulus required to maintain bone homeostasis, partly through the renewal and osteogenic differentiation of mesenchymal stem cells (MSCs). However, the mechanism by which MSCs sense a biophysical force and translate that into a biochemical bone forming response (mechanotransduction) remains poorly understood. The primary cilium is a single sensory cellular extension, which has recently been shown to demonstrate a role in cellular mechanotransduction and MSC lineage commitment. In this study, we present evidence that short periods of mechanical stimulation in the form of oscillatory fluid flow (OFF) is sufficient to enhance osteogenic gene expression and proliferation of human MSCs (hMSCs). Furthermore, we demonstrate that the cilium mediates fluid flow mechanotransduction in hMSCs by maintaining OFF-induced increases in osteogenic gene expression and, surprisingly, to limit OFF-induced increases in proliferation. These data therefore demonstrate a pro-osteogenic mechanosensory role for the primary cilium, establishing a novel mechanotransduction mechanism in hMSCs. Based on these findings, the application of OFF may be a beneficial component of bioreactor-based strategies to form bone-like tissues suitable for regenerative medicine and also highlights the cilium as a potential therapeutic target for efforts to mimic loading with the aim of preventing bone loss during diseases such as osteoporosis. Furthermore, this study demonstrates a role for the cilium in controlling mechanically mediated increases in the proliferation of hMSCs, which parallels proposed models of polycystic kidney disease. Unraveling the mechanisms leading to rapid proliferation of mechanically stimulated MSCs with defective cilia could provide significant insights regarding ciliopathies and cystic diseases. Stem Cells2012;30:2561–2570