Topographical and mechanical cues are vital for cell fate, tissue development in vivo, and to mimic the native cell growth environment in vitro. To date, the combinatory effect of mechanical and topographical cues as not been thoroughly investigated. This study investigates the effect of PCL nanofiber alignment and hydrostatic pressure on stem cell differentiation for bone tissue regeneration. Bone marrow‐derived human mesenchymal stem cells were seeded onto standard tissue culture plastic and electrospun random and aligned nanofibers. These substrates were either cultured statically or subjected to intermittent hydrostatic pressure at 270 kPa, 1 Hz for 60 min daily over 21 days in osteogenic medium. Data revealed higher cell metabolic activities for all mechanically stimulated cell culture formats compared with non‐stimulated controls; and random fibers compared with aligned fibers. Fiber orientation influenced cell morphology and patterns of calcium deposition. Significant up‐regulation of Collagen‐I, ALP, and Runx‐2 were observed for random and aligned fibers following mechanical stimulation; highest levels of osteogenic markers were expressed when hydrostatic pressure was applied to random fibers. These results indicate that fiber alignment and hydrostatic pressure direct stem cell fate and are important stimulus for tissue regeneration. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: A: 629–640, 2018.