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We prepared a three-dimensional cell culture system using atelocollagen gel derived from chum salmon sources. To examine whether collagen gel derived from salmon is suitable as a scaffold for ES-D3 cell differentiation, we compared the degrees of differentiation of cells cultured in a salmon collagen (FC), a conventional collagen gel (CG), and a collagen sponge derived from porcine sources (CS). No significant differences in cell differentiation were noted between cells cultured with the collagen gel derived from salmon and the control cells, the differentiation of which was inhibited by adding mLIF. With FC, efficient differentiation of ES-D3 cells was not observed, whereas both the collagen gel supported cell differentiation and the collagen sponge derived from the porcine sources. The results of studies using ES cells have proven useful in studies of ES cell differentiation factors. Moreover, these study results may yield important clues as to cell differentiation factors for adult stem cells. To that end, the development of scaffolds that facilitate the in vitro culturing of ES cells for long periods is desirable.