Here we describe a protocol for generating 3D\u00a0human\u00a0intestinal\u00a0tissues (called\u00a0organoids) in vitro from\u00a0humanpluripotent stem cells (hPSCs). To generate\u00a0intestinal\u00a0organoids, pluripotent stem cells are first differentiated into FOXA2(+)SOX17(+) endoderm by treating the cells with activin A for 3 d. After endoderm induction, the pluripotent stem cells are patterned into CDX2(+) mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step, 3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors, and they proliferate and expand over 1-3 months to give rise to\u00a0intestinal\u00a0tissue, complete with\u00a0intestinal\u00a0mesenchyme and epithelium comprising all of the major\u00a0intestinal\u00a0cell types. To date, this is the only method for efficiently directing the differentiation of hPSCs into 3D\u00a0human\u00a0intestinal\u00a0tissue in vitro.