Efficient production of definitive endoderm from embryonic stem (ES) cells opens doors to the possibilities of differentiation of endoderm-derived tissues such as the intestines, pancreas, and liver that could address the needs of people with chronic diseases involving these organs. The lessons learned from developmental biology have contributed significantly to in vitro differentiation of definitive endoderm. Gastrulation, a process that results in the production of all three embryonic germ cell layers, definitive endoderm, mesoderm, and ectoderm, is an important step in embryonic development. Gastrulation occurs as a result of the events that are orchestrated by the signaling pathways involving Nodal, FGF, Wnt, and BMP. Understanding these signaling pathways has led to the introduction of key ingredients such as Activin A, FGF, Wnt, and BMP to the differentiation protocols that have been able to produce definitive endoderm from ES cells. Efficient production of definitive endoderm needs to meet the specific criteria that include (a) increase in the production of markers of definitive endoderm such as Sox 17, FOXA2, GSC, and Mixl1; (b) decrease in the production of markers of primitive/visceral/parietal endoderm, Sox 7 and OCT4; and (c) decrease in the mesoderm markers (Brachyury, MEOX) and ectoderm markers (Sox1 and ZIC1).