Human mesenchymal stem cells (hMSCs) have the potential to differentiate into different types of mesodermal tissues. In vitro proliferation and differentiation of hMSCs are necessary for bone regeneration in tissue engineering. The present study aimed to design and develop a fluid flow mechanically-assisted cartridge device to enhance the osteogenic differentiation of hMSCs. We used the fluorescence-activated cell-sorting method to analyze the multipotent properties of hMSCs and found that the cultured cells retained their stemness potential. We also evaluated the cell viabilities of the cultured cells via water-soluble tetrazolium salt 1 (WST-1) assay under different rates of flow (0.035, 0.21, and 0.35 mL/min) and static conditions and found that the cell growth rate was approximately 12% higher in the 0.035 mL/min flow condition than the other conditions. Moreover, the cultured cells were healthy and adhered properly to the culture substrate. Enhanced mineralization and alkaline phosphatase activity were also observed under different perfusion conditions compared to the static conditions, indicating that the applied conditions play important roles in the proliferation and differentiation of hMSCs. Furthermore, we determined the expression levels of osteogenesis-related genes, including the runt-related protein 2 (Runx2), collagen type I (Col1), osteopontin (OPN), and osteocalcin (OCN), under various perfusion vis-à-vis static conditions and found that they were significantly affected by the applied conditions. Furthermore, the fluorescence intensities of OCN and OPN osteogenic gene markers were found to be enhanced in the 0.035 mL/min flow condition compared to the control, indicating that it was a suitable condition for osteogenic differentiation. Taken together, the findings of this study reveal that the developed cartridge device promotes the proliferation and differentiation of hMSCs and can potentially be used in the field of tissue engineering.