The adult colonic epithelium performs distinct physiological functions such as presenting a mucosal barrier to microorganisms and regulating the immune response to pathogens. Although there are various reports using animal models and cell lines to study colonic immunity and inflammatory and infectious diseases, they cannot thoroughly recapitulate the human colon in vivo. This issue has been addressed by developing methods to generate in vitro primary colon organoids from human colon tissue samples. Certain limitations such as limited supply can be overcome by generating colonic tissues from induced pluripotent stem cells (iPSC). Some studies have been reported so far that sought to develop effective protocols for deriving colonic organoids from human iPSC (Crespo et al., 2017; Múnera et al., 2017). With the aim of modelling human large intestine, we set up a stepwise, defined, serum-free and 38-day protocol for the progressive generation of definitive endoderm, mid-hind gut endoderm, hind gut endoderm, and subsequently, colon organoids through the modifications of some recently-published papers. A cocktail of embryonic stage-specific growth factors was applied to induce signalling pathways which were required to be modulated in each step. Flow cytometry analysis at day 3 of differentiation confirmed that around 100% of iPSCs were converted into definitive endoderm which is the main step in the endoderm differentiation. Extensive immunohistochemical analysis also proved that the 38-day iPSC-derived colon organoids mimic human colon epithelium through the expression of colon-specific markers (SATB2 and MUC5B), gut-specific markers (MUC2, CHGA and CDX2), junction and polarity-related markers (Zo-1, Villin and E-cadherin). iPSC-derived gut epithelium also secreted interferon epsilon, an epithelial cytokine under study in our lab in the control of intestinal infections. We are currently characterising the pattern recognition receptor pathway responses of iPSC-derived colonic epithelium to micro-organisms and synthetic innate immune stimulating ligands. In conclusion, our study provides an efficient method for differentiating human colon organoids from iPSCs, which have the advantage of relatively unlimited supply, ability to genetically manipulate (gene expression, etc), could study interactions with “matched” (e.g., immune) cell types, and can “capture” genetic of an individual such as modifications in “susceptibility genes”.