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Definitive endoderm (DE) derived from human pluripotent stem cells (hPSCs) holds great promise for cell-based therapies and drug discovery. However, current DE differentiation methods required undefined components and/or expensive recombinant proteins, limiting their scalable manufacture and clinical use. Homogeneous DE differentiation in defined and recombinant protein-free conditions remains a major challenge. Here, by systematic optimization and high-throughput screening, we report a chemically defined, small-molecule-based defined system that contains only four components (4C), enabling highly efficient and cost-effective DE specification of hPSCs in the absence of recombinant proteins. 4C-induced DE can differentiate into functional hepatocytes, lung epithelium, and pancreatic β cells in vitro and multiple DE derivatives in vivo. Genomic accessibility analysis reveals that 4C reconfigures chromatin architecture to allow key DE transcription factor binding while identifying TEAD3 as a novel key regulator of the process. This system may facilitate mass production of DE derivatives for drug discovery, disease modeling, and cell therapy., Competing Interests: Declaration of interests N.C., Z.Z., and F.Z. have filed patent applications related to DE differentiation by 4C., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)