Human embryonic germ cell derivatives, a heterogeneous population of uncommitted embryoid body derived (EBD) cells, were studied in a bioactive three-dimensional (3D) fibrous culture. Their proliferation, morphology, gene expression and differentiation were investigated to gain insights on development of 3D bioactive scaffold for pluripotent stem cells. The expansion of the EBD cells in 3D environment was significantly higher than their two-dimensional controls after 21 days. No apparent differentiation of the EBD cells cultured in the 3D environment, as indicated by histology and gene expression profile analysis, was evident. Extracellular matrix production was weak in the long-term 3D culture, and the EBD cells maintained their multilineage gene expressions for the period studied. When nerve growth factor (NGF) was surface-immobilized on the fibrous scaffold via chemically-modified Pluronic, the EBD cells cultured in this scaffold showed evidence of entering the neural pathway. An upregulation of tyrosine hydroxylase mRNA expression was observed when EBD cells were cultured in the NGF-immobilized fibrous scaffold, as demonstrated by real-time PCR and immunofluorescence staining. The study suggests the value of such fibrous 3D culture in manipulating stem cell proliferation/differentiation and as a model for developing a bioactive scaffold.