Dopaminergic differentiation of embryonic stem cells (ESCs) gains more and more attention worldwide owing to its potential use for neurorestorative therapy for the treatment of Parkinson's disease. The conventional 2D cell culture on petri dishes with various animal derived substrata such as collagen gels, laminin, and Matrigel is widely used to induce dopaminergic differentiation and it may limit the efficiency in the generation of dopaminergic neurons from ESCs and prevent their application for human therapies. Here, we reported that a self-assembling peptide made from natural amino acids has a property to generate a true 3D environment for dopaminergic differentiation. Mouse ESCs (R1) and mouse iPSCs (TTF-1) embedded in RADA16-I peptide-derived nanofiber scaffolds led to a marked increase in dopaminergic differentiation compared to the laminin-coated 2D culture or Matrigel-encapsulated 3D culture. These differentiated neurons expressed specific dopaminergic markers and produced appropriate patterns of action potential firing. Consistent with the increase in the number of dopaminergic neurons differentiated from R1 or TTF-1 in the self-assembling peptide nanofiber scaffold (SAPNS), both the expression levels of genes that involve in dopaminergic differentiation and maturation and the dopamine release in SAPNS culture were significantly elevated. The results of the study suggest that SAPNS provides a promising 3D culture system for dopaminergic differentiation. © 2013 Ni et al.