We establish in utero lentiviral transduction as a powerful approach for tracing cell lineages and manipulating gene function in the developing gut. We find that mesenchymal cells acquire positional and fate restrictions early, whereas the enteric nervous system retains broad developmental competence and gains regional identities later. We also show clonal linkage between specific fibroblast subtypes and more specialised mesenchymal cell types that would be difficult resolve without the barcode-based lineage tracing approach. This framework reveals fundamental principles of gut organogenesis and provides new opportunities to study the developmental origins of visceral dysfunction.