Human embryonic stem cells (hESCs) have the capacity to self-renew and to differentiate into all components of the embryonic germ layers (ectoderm, mesoderm, endoderm) and subsequently all cell types that comprise human tissues. HESCs can potentially provide an extraordinary source of cells for tissue engineering and great insight into early embryonic development. Much attention has been given to the possibility that hESCs and their derivatives may someday play major roles in the study of the development, disease therapeutics, and repair of injuries to the central and peripheral nervous systems. This tantalizing promise will be realized only when we understand fundamental biological questions about stem cell growth and development into distinct tissue types. In vitro, differentiation of hESCs into neurons proceeds as a multistep process that in many ways recapitulates development of embryonic neurons. We have found in vitro conditions that promote differentiation of stem cells into neuronal precursor or neuronal progenitor cells. Specifically, we have investigated the ability of two federally approved hESC lines, HSF-6 and H7, to form embryonic and mature neuronal cells in culture. Undifferentiated hESCs stain positively for markers of undifferentiated/pluripotent hESCs including surface glycoproteins, SSEA-3 and 4, and transcription factors Oct-3/4 and Nanog. Using reduced numbers of mouse embryonic fibroblasts as feeder substrates, these markers of pluripotency are lost quickly and replaced by primarily neuroglial phenotypes with only a few cells representing other embryonic germ layer types remaining. Within the first 2 weeks of co-culture with reduced MEFs, the undifferentiated hESCs show progression from neuroectodermal to neural stem cell to maturing and migrating neurons to mature neurons in a stepwise fashion that is dependent on both the type of hESCs and the density of MEFs. In this chapter, we provide the methods for culturing pluripotent hESCs and MEFs, differentiating hESCs using reduced density MEFs, and phenotypic analyses of this culture system.