PROJECT SUMMARY Organogenesis is a process in which biochemical signals and mechanical cues transform embryonic germ layers into organs during fetal development. Recent advances in stem cell biology created opportunities to differenti- ating human pluripotent stem cells into expandable 3D tissues that contain many of the cellular and functional characteristics of fetal organs, which we call organoids. They hold a tremendous potential to answer longstand- ing questions of human development and one day to serve as a renewable source of patient-specific tissues. However, organoids still only approximatively recapitulate organogenesis as they rely on spontaneous tissue self-organization and the establishment of signaling gradients in unpredictable ways. Common protocols also neglect the contribution of surrounding developing organs, especially from other germ layers, and because they lack the vasculature, they are not scalable. This supplement proposal aims precisely to advance these gaps in the current organoid paradigm; namely, by generating organoids from all three germ layers and by mimicking in vitro angiogenesis to increase their scalability. We focus on the organogenesis of the gut tube, the embryonic structure on which many adjacent organs form, and whose axial patterning strongly depends on the signaling feedback between all three germ layers. We propose to employ microfluidics to generate precise signaling gra- dients so to reproducibly mimic both the signaling and the morphogenesis of gut tube organogenesis in vitro. We will generate carefully assembled components of the primitive gut tube comprising all three germ layers, then expose them to signaling gradients in a highly controlled mechanical environment. Combined with live- cell microscopy, CRISPR editing, and single cell transcriptomics, our multi-organoids will reveal a detailed hi- erarchy of fate choices cells make from pluripotency to regionally segmented gut tube, and they will allow us to make crucial connections between signaling, transcriptional regulation, and tissue morphogenesis. Our work will shed light on the largely unknown regulatory mechanisms by which the three germ layers communicate to generate complex and asymmetric patterns along the body axes.