Extracellular environment regulates cell behavior and also influences the differentiation of stem cells. Two cell lines of pluriopotent human embryonic germ cell derivatives (EBD cells) were cultured on a biodegradable poly(epsilon-caprolactone-co-ethyl ethylene phosphate) (PCLEEP) and non-degradable cellulose acetate scaffold. Their cell behaviors including proliferation, differentiation, cell distribution and extracellular matrix production were studied for 4 weeks and 10 months. The proliferation of the EBD cells was enhanced in both of the three-dimensional scaffolds in the first 5 weeks of culture, regardless of the material difference, compared to monolayer culture. While the gene expression profile remained multilineage for the EBD cells cultured in the cellulose acetate fibrous scaffold, much of the neuronal lineage markers were down-regulated in EBD cells cultured in the PCLEEP scaffold. On the other hand, extracellular matrix production was significantly enhanced in the PCLEEP scaffold. The study showed that the polymer substrate could influence the differentiation and growth of pluripotent stem cells in the absence of exogenous biochemical signals.