A method has been developed to efficiently direct the differentiation of human pluripotent stem cells (PSCs) into intestinal tissue in vitro. This process involves a series of growth factor manipulations to mimic embryonic intestinal development. The differentiation begins with the formation of definitive endoderm (DE) using ActivinA, followed by the specification of posterior endoderm and hindgut development using FGF4 and Wnt3a. The resulting three-dimensional intestinal "organoids" consist of a polarized, columnar epithelium with villus-like structures and crypt-like proliferative zones that express intestinal stem cell markers. These organoids contain functional enterocytes, goblet, Paneth, and enteroendocrine cells. The study identified that the combined activity of Wnt3a and FGF4 is required for hindgut specification, while FGF4 alone is sufficient for hindgut morphogenesis. The research also demonstrated that NEUROG3, a pro-endocrine transcription factor, is necessary and sufficient for human enteroendocrine cell development in vitro. The organoids were able to mature in vitro, developing intestinal stem cells and acquiring both absorptive and secretory functionality. This system allows for functional studies of human congenital gut defects and the generation of intestinal tissue for potential transplantation-based therapies. The method is broadly applicable to various PSC lines, including both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). The organoids were able to expand significantly over time, with a cellular gain of up to 1,800-fold. The study highlights the potential of PSC-derived intestinal tissue for understanding human intestinal development and disease.A method has been developed to efficiently direct the differentiation of human pluripotent stem cells (PSCs) into intestinal tissue in vitro. This process involves a series of growth factor manipulations to mimic embryonic intestinal development. The differentiation begins with the formation of definitive endoderm (DE) using ActivinA, followed by the specification of posterior endoderm and hindgut development using FGF4 and Wnt3a. The resulting three-dimensional intestinal "organoids" consist of a polarized, columnar epithelium with villus-like structures and crypt-like proliferative zones that express intestinal stem cell markers. These organoids contain functional enterocytes, goblet, Paneth, and enteroendocrine cells. The study identified that the combined activity of Wnt3a and FGF4 is required for hindgut specification, while FGF4 alone is sufficient for hindgut morphogenesis. The research also demonstrated that NEUROG3, a pro-endocrine transcription factor, is necessary and sufficient for human enteroendocrine cell development in vitro. The organoids were able to mature in vitro, developing intestinal stem cells and acquiring both absorptive and secretory functionality. This system allows for functional studies of human congenital gut defects and the generation of intestinal tissue for potential transplantation-based therapies. The method is broadly applicable to various PSC lines, including both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). The organoids were able to expand significantly over time, with a cellular gain of up to 1,800-fold. The study highlights the potential of PSC-derived intestinal tissue for understanding human intestinal development and disease.