Neural crest cells (NCCs) arise between neural and non-neural ectoderm and migrate to form a variety of NC derivatives (1,2). Vertebrate NCCs represent a somatic cell type with unique properties with pluripotency and capable of various cell fates (3-6). However, despite extensive studies across many animal species, our understanding of human NC development remains limited. Defects in human NC development are responsible for medical problems such as Hirschsprung’s disease, DiGeorge syndrome, Waardenburg syndrome, Charcot-Marie-Tooth disease, Familial Dysautonomia and pediatric cancers such as neuroblastoma (7-12). For better understanding of NC development, it will be essential to have access to NCCs in a in vitro model system. Since the isolation of human embryonic stem cells (hESC) by Thomson group, hESCs have become a valuable for early human development (13). Neural induction in hESCs is characterized by the formation of neural rosettes, representing the early developing neuroepithelium (14-18). Neural rosettes can give rise to cells of NC and be used for isolation of NC populations. The use of hESC-NCCs serves as a platform for understanding NC development and disorders. The protocol has been validated in different hESCs and hiPSC lines. The average time required for NC precursors using this protocol is 4-5 weeks.