Induced pluripotent cells have been considered as one of the most recent and best cell sources for the cell therapy. In this study, the differentiation potency of human iPS cells, cultured on scaffolds, which can differentiate into definitive endodermal cells as precursor for hepatocytes, pancreatic and lung cells, was studied. Embryoid bodies composed of pluripotent cells, were seeded on electrospinning nanofiber scaffold. The cells were differentiated into definitive endoderm using IDE1. Expression of definitive endoderm markers including Sox17, FoxA2 and GSC were confirmed by immunocytochemistry staining and qRT-PCR analysis. In the present study, morphology and viability of cells were evaluated by utilizing a scanning electron microscopy and MTT assay, respectively. The results demonstrated the positive effect of 3D cultures, using suitable factors, on definitive endoderm differentiation.