In this study, we developed multi-functional biomimetic tissue engineered scaffolds that are capable of controlling the spatial locations of stem cells and releasing multiple growth factors with a controlled dose and rate of delivery. This novel scaffold was fabricated by combining electrospinning and photolithography and consisted of polycaprolactone (PCL)/gelatin fibers and poly(ethylene glycol) (PEG) hydrogel micropatterns. The utility of this system was investigated in the context of the osteogenesis of human mesenchymal stem cells (hMSCs). When hMSCs were seeded onto hydrogel-incorporated fibrous scaffolds, they selectively adhered and grew only in the fiber region due to the non-adhesiveness of the PEG hydrogel, enabling spatial positioning of hMSCs on a micrometer scale. For osteogenic differentiation of hMSCs, basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) were loaded on the fibers and within the hydrogel matrix, respectively, to enable sequential delivery of low doses of bFGF during the early stages and sustained release of BMP-2 for long periods. According to in vitro studies, hMSCs cultured on the scaffolds capable of sequential delivery of bFGF and BMP-2 showed stronger osteogenic commitment in culture than those on scaffolds without any growth factors or scaffolds with single administration of either bFGF or BMP-2 under the same conditions. The results demonstrate that hydrogel-incorporated fibrous scaffolds can provide not only biomimetic structures with micropatterned nanostructures but also a suitable biochemical environment with controlled release of multiple growth factors, which may eventually facilitate the control of stem cell fates for various regenerative therapies.