Human intestinal organoids (HIOs) derived from pluripotent stem cells are valuable tools for studying gastrointestinal development and function. Herein, we present a method for generating and co-culturing human intestinal organoids and enteric glial cells derived from human pluripotent stem cells. In addition, we describe how to collect and stain enteric glial cells within organoids embedded in Matrigel. The method outlines the precise steps required to successfully generate both human intestinal organoids and vagal neural crest cells, offering valuable insight into the study of human enteric glial cells.