Abstract: Parasympathetic neurons (parasymN) are important for unconscious body responses, including rest-and-digest and calming the body. Disclosed herein is a chemically defined differentiation protocol that generates parasympathetic neurons from stem cells. The protocol yields high efficiency and purity cultures that are electrically active and respond to specific stimuli. Their molecular characteristics and maturation stage are described and evidence for their use as a model for studying parasymN function and dysfunction. Cell populations and compositions formed from the resulting cells, as well as methods of their use for disease treatment, drug screening, and modeling of human disorders affecting parasymN are also provided.