Human induced pluripotent stem cells (hiPSCs) are attractive sources of cells for disease modeling in vitro, and they may eventually provide access to cells/tissues for the treatment of many degenerative diseases. Stepwise differentiation from hiPSCs to definitive endoderm (DE) will identify a key step in hepatocytes and beta cell development and may prove useful for transplantation therapy for liver diseases and diabetes. Inducer of definitive endoderm 1 (IDE1) is known to play an important role in the regional specification of DE. Here, we have investigated the effect of stimulation with IDE1 on the development of hiPSCs into DE cells in three-dimensional (3D) cultures. The differentiation was determined by immunofluorescence staining with Sox17, FoxA2, and goosecoid (Gsc) and also by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis. In this study, we showed that hiPSCs with 6-day IDE1 treatment (as chemical tool) on poly(epsilon-caprolactone) (PCL) nanofibrous scaffold were able to differentiate into DE cells.