Flow-perfusion is being promoted as a way to grow tissue-engineered cartilage in vitro. Yet, there is a concern that flow-perfusion may induce unwanted mechanical effects on chondrogenesis and terminal differentiation. Therefore, the aim of this study is to evaluate the effect of fluid flow on chondrogenesis and chondrocyte hypertrophy of MSCs in a well-established pellet culture model.Human MSC pellets were mounted into 3D-printed porous scaffolds in basic chondrogenic differentiation medium, containing TGF-ß2. Constructs were then allowed to form cartilaginous matrix for 18 days, before they were transferred to a custom-built flow-perfusion system. A continuous flow of 1.22mlmin-1 was applied to the constructs for 10 days. Controls were maintained under static culture conditions. To evaluate chondrogenic and hypertrophic differentiation, RNA was isolated at day 20 and 28 and histology, immunohistochemistry and western blot analyses were performed after 28 days of culture.Abundant matrix was formed in the constructs, but production of chondrogenic and hypertrophic matrix components was affected by flow-perfusion. Although gene expression levels of the (late) hypertrophic and osteogenic marker osteocalcin increased by flow-perfusion, this did not result in more collagen type X protein deposition. Decreased GAG release, in combination with diminished collagen II staining, indicates reduced chondrogenesis in response to flow-perfusion.Caution should thus be taken when applying flow-perfusion to cultures to improve nutrient diffusion. Although we show that it is possible to influence the differentiation of chondrogenic differentiated MSCs by flow-perfusion, effects are inconsistent and strongly donor-dependent. © 2013 Elsevier Ltd.