The way to overcome current limitations in the generation of glucose-responsive insulin-producing cells is selective enrichment of the number of definitive endoderm (DE) progenitor cells. Sox17 is the marker of mesendoderm and definitive endoderm. The aim of the present research was to study the potential of Sox17 knock-in CGR8 mouse embryonic stem (ES) cells to differentiate into insulin producing cells in vitro by detection of specific markers at different stages of three-step protocol of differentiation. At day 5+9 cells strongly expressed Sox17 as well as CxCr4 and N-cadherine, while staining for Oct-4 (octamer-binding transcription factor 4) was faint. At day 5+16 the expression of Sox17 gradually disappeared and cells were positive for Neurogenin 3 (NGN3), Islet-1 and C-peptide. At last stage of differentiation protocol (day 5+23) Sox17 knock-in CGR8 ES derived cells showed insulin and C-peptide expression. We conclude that Sox17 knock-in mouse ES cells are equally suitable for differentiation into insulin producing cells as commonly used CGR8 ES cells. Genetically modified ES cells allow the selection of Sox17-expressing cells and the selective enrichment of DE progenitors. Manuscript profile