Extensive expansion of mesenchymal stem cells (MSCs) for cell-based therapies remains challenging since long-term cultivation and excessive passaging in two-dimensional conditions result in a loss of essential stem cell properties. Indeed, low survival rate of cells, alteration of surface marker profiles, and reduced differentiation capacity are observed after in vitro expansion and reduce therapeutic success in clinical studies. Remarkably, cultivation of MSCs in three-dimensional aggregates preserve stem cell properties. Hence, the large scale formation and cultivation of MSC aggregates is highly desirable. Besides other effects, MSCs cultivated under hypoxic conditions are known to display increased proliferation and genetic stability. Therefore, in this study we demonstrate cultivation of adipose derived human MSC aggregates in a stirred tank reactor under hypoxic conditions. Although aggregates were exposed to comparatively high average shear stress of 0.2 Pa as estimated by computational fluid dynamics, MSCs displayed a viability of 78–86% and maintained their surface marker profile and differentiation potential after cultivation. We postulate that cultivation of 3D MSC aggregates in stirred tank reactors is valuable for large-scale production of MSCs or their secreted compounds after further optimization of cultivation parameters.