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Definitive endoderm (DE) is the first stage of human pluripotent stem cell (hPSC) differentiation into hepatocyte-like cells. Developing human liver cell models for pharmaceutical applications is highly demanding. Due to the vast number of existing protocols to generate DE cells from hPSCs, we aimed to compare the specificity and efficiency of selected published differentiation conditions. We differentiated two hPSC lines (induced PSC and embryonic stem cell) to DE cells on Matrigel matrix using growth factors (ActivinAandWnt-3a) and smallmolecules (sodiumbutyrate and IDE1) in different combinations. By studying dynamic changes during 6 days in cell morphology and the expression of markers for pluripotency, DE, and other germ layer lineages, we found that Activin A is essential for DE differentiation, while Wnt-3a and sodium butyrate are dispensable. Although sodium butyrate exerted rapidDE differentiation kinetics, it caused massive cell death and could not generate sufficient cells for further differentiation and applications.We further discover that IDE 1 could not induce DE as reported previously. Hereby, we compared different conditions for DE induction and found an effective six day-protocol to obtain DE cells for the further differentiation and applications. [ABSTRACT FROM AUTHOR]