Patterning of the gastrointestinal tract during development is crucial for proper gut function, and understanding the mechanisms underpinning this process is an active area of research. RFX6 is a transcription factor that has been implicated in the development of various gastrointestinal organs, including the proximal region of the small intestine (the duodenum). Here, James Wells and colleagues generate human intestinal organoids from a person presenting with duodenal defects and carrying a compound heterozygous RFX6 mutation. The organoids exhibit reduced levels of PDX1, a transcription factor that drives duodenal gene expression, and have inappropriate expression of gastric and ileal markers. These indicators of disrupted duodenal identity are also observed in biopsies from the individual’s duodenum. Correcting the RFX6 mutation using CRISPR/Cas9 largely restores proper duodenal patterning in the organoids, including restoration of PDX1 expression. Re-introduction of PDX1 expression in the organoids can also rescue some aspects of duodenal patterning, and comparison of genes that are activated by either RFX6 or PDX1 induction suggests that RFX6 sits upstream of PDX1. The authors take a bioinformatics approach to identify a pdx1 enhancer and use luciferase assays in the Xenopus model system to show that Rfx6 is required to activate this enhancer. Overall, this study leverages organoids to uncover novel disease phenotypes caused by disruption of RFX6 and identifies that establishment of duodenal identity in vertebrates requires RFX6 activation of PDX1.