Taking tissue engineering applications into clinical trials requires the development of efficient and safe protocols incorporated with effective 3-dimensional cell culturing and differentiation systems in order to develop transplantable tissues that may offer a life-line for patients in the future. Cord blood, which is perhaps the most abundant world stem cell source, has shown previously practical and ethical advantages over other stem cells sources in many research and clinical applications including regenerative medicine. We previously developed a three-step protocol for isolation, expansion and sequential neuronal differentiation of cord blood pluripotent stem cells (characterized with our unique triple immunocytochemisty scheme for Oct-4, Sox-2 and Nanog) in defined serum-free culturing conditions. In this study we incorporated this protocol with 3-dimensional culturing systems which produced artificial neuronal tissues expressing Nestin, NF-200, TUJ1, PSD-95 and NeuN. We showed that cord blood pluripotent stem cells are a potential and promising candidate for future neural tissue engineering and regenerative medicine.