Human Mesenchymal Stromal Cells (hMSCs) are a safe option for allogeneic cell therapy across various diseases, but their manufacturing process requires improvement to broaden accessibility. In this study, a state-of-the-art planar multi-vessel process was transferred to a stirred tank bioreactor using microcarriers to support the growth of adherent cells. The frequent medium exchange strategy from planar culture guided the design of the bioreactor process. However, complete medium changes in the bioreactor resulted in limited cell expansion and higher glucose consumption compared to planar culture. To enhance expansion, a 0.1 L perfusion bioreactor was tested, enabling continuous medium exchange. Three perfusion approaches were evaluated: (1) maintaining a target cell-specific glucose consumption rate, (2) varying the perfusion rate, and (3) applying a cell-specific perfusion rate. Implementing targeted glucose feeding (TAFE) reduced lactate production, while increasing perfusion rates improved cell density. The highest expansion was achieved using a cell-specific perfusion rate of 5 nL cell- 1 day- 1 combined with a target glucose consumption rate (qglc) of 15 pmol cell- 1 day- 1, resulting in a 5.4-fold higher expansion factor than daily medium changes in stirred tank bioreactors. This optimized process represents a key advancement toward producing clinically relevant quantities of hMSCs.