Perfusion bioreactor systems play a crucial role in mitigating nutrient limitation as well as providing biomechanical stimuli and redistributing regulatory macromolecules that influence human mesenchymal stem cells (hMSC) fate in three-dimensional (3D) scaffolds. As fibroblast growth factor-2 (FGF-2) is known to regulate hMSC phenotype, understanding the role of autocrine FGF-2 signaling in the 3D construct under the different perfusion flow provides important insight into an optimal bioreactor design. To investigate FGF-2 signaling inhibition in hMSC cultured in the porous poly(ethylene terephthalate) (PET) scaffolds perfused under two flow configurations, PD173074, an FGFR1 inhibitor, was added in growth media after 7 day of pre-culture and its impact on hMSC proliferation and clonogenicity during the subsequent 7 days of cultivation was analyzed. Compared with control constructs in growth media, the addition of PD173074 resulted in significant reduction in hMSC proliferation and colony formation in both constructs with a more dramatic reduction in the parallel flow constructs. The results demonstrate that autocrine FGF-2 plays a significant role in 3D scaffold and suggest modulation of the perfusion flow in the bioreactor as a strategy to influence autocrine actions and cell fate in the 3D scaffold. (c) 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012