Neural stem cells (NSC) are not only a valuable tool for the study of neural development and function, but an integral component in the development of transplantation strategies for neural disease. NSC can be used to study how neurons acquire distinct phenotypes and how the reciprocal interactions between neurons and glia in the developing nervous system shape the structure and function of the central nervous system (CNS). In addition, neurons prepared from NSC can be used to elucidate the molecular basis of neurological disorders as well as potential treatments. Although NSC can be derived from different species and many sources, including embryonic stem cells, induced pluripotent stem cells, adult CNS, and direct reprogramming of non-neural cells, isolating primary NSC directly from rat fetal tissue is the most common technique for preparation and study of neurons with a wealth of data available for comparison. Regardless of the source material, similar techniques are used to maintain NSC in culture and to differentiate NSC toward mature neural lineages. This chapter will describe specific methods for isolating multipotent NSC and neural precursor cells (NPC) from embryonic rat CNS tissue (mostly spinal cord). In particular, NPC can be separated into neuronal and glial restricted precursors (NRP and GRP, respectively) and used to reliably produce neurons or glial cells both in vitro and following transplantation into the adult CNS. This chapter will describe in detail the methods required for the isolation, propagation, storage, and differentiation of NSC and NPC isolated from rat spinal cords for subsequent in vitro or in vivo studies.