Development of the enteric nervous system (ENS) begins early in embryonic development with the rostrocaudal migration of the neuronal precursors, enteric neural crest-derived cells (ENCDCs), to the gut. ENCDCs then differentiate into the myenteric and submucosal plexuses of the ENS. While genetic factors direct appropriate ENCDC migration and differentiation, recent evidence of disrupted function and architecture of the myenteric plexus in germ-free (GF) mice in comparison to control specific pathogen free (SPF) mice suggests that intestinal microbiota may also play an important role. This study aimed to establish an in vitro ENCDC model as a platform for assessing microbiota-ENS interactions. SPF fetal guts (n=48) were harvested at embryonic day 15. ENCDCs were selected using magnetically-activated cell sorting and were cultured in neuronal cell media. Staining with antibodies against ENCDC marker p75, neuronal marker HuC/D, and nuclear marker bisbenzimide showed 99.1% of cells to be p75 positive. Antibodies against serotonin, neuronal nitric oxide synthase, tyrosine hydroxylase, and proliferation marker phospho-histone H3 uncovered 1.57% serotonergic neurons, 5.72% nitrergic neurons, 0% dopaminergic neurons, and 4.33% proliferating cells. This suggested maintenance of ENCDCs in their undifferentiated state across subcultures. Future work will involve incubation of this established ENCDC cell line with varying concentrations of microbial products. Proportions of neurotransmitter subtypes will be compared to the established controls to determine the effects of microbial products on ENCDC differentiation. This research may have implications for the impact of disturbing perinatal microbiota, whether via antibiotic administration or maternal diet modification, on perinatal development of the ENS.