Migration of enteric neural crest cells (ENCCs) within the developing embryonic gut is key to the formation of the enteric nervous system (ENS). Investigating factors that guide the migration of these cells along the gut tube could aid in understanding the pathogenesis and developing potential therapy for ENS developmental defects, such as those present in Hirschsprung disease. However, limitations exist on appropriate study models to allow the live tracing of these migrating cells within the developing intestine. This protocol describes a mouse embryonic gut explant model utilizing a Sox10-Venus transgenic mouse to fluorescently label migrating ENCCs within the gut for live-cell imaging during culturing.