Human embryonic stem cell (hESC)-derived endodermal cells are of interest for the development of cellular therapies to treat disorders such as liver failure. The soluble form of activin A (Act) has been widely used as an in vitro inducer of definitive endoderm (DE). In this study, we have developed a nanofibrous poly (E-caprolactone) substrate, biofunctionalized with Act, for directed differentiation of hESCs into DE. Bioconjugation of Act on nanofibrous meshes was confirmed by enzyme-linked immunosorbent assay (ELISA) and immunostaining. In order to investigate the bioactivity of immobilized Act (iAct), hESCs were cultivated on the Act-conjugated nanofibers for five days. The nanofibers with covalent iAct significantly increased expression levels of the endodermal markers SOX17, FOXA2, and CXCR4, compared with physically adsorbed Act (aAct) or without Act (noAct). In addition, iAct retained its bioactivity after storage for five days in the absence of cell seeding. The capability of cultivated cells to generate the DE-derived lineage was evaluated through further differentiation of seeded cells into hepatocyte-like cells (HLCs). Interestingly, the iAct sample showed a higher level of hepatic markers compared to the aAct sample. We also demonstrated that iAct in the presence of soluble Act (sAct) could improve the conventional protocol to generate HLCs from hESCs. (C) 2016 Wiley Periodicals, Inc.