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Background: Coating a culture plate with molecules that aid in cell adhesion is a technique widely used to produce animal cell cultures. Extracellular matrix (ECM) is known for its efficiency in promoting adhesion, survival, and proliferation of adherent cells. Gelatin, a cost-effective type of ECM, is widely used in animal cell cultures including feeder-free embryonic stem (ES) cells. However, the optimal concentration of gelatin is a point of debate among researchers, with no studies having established the optimal gelatin concentration. Methods: In this study, we coated plastic plates with gelatin in a concentrationdependent manner and assessed Young’s modulus using atomic force microscopy (AFM) to investigate the microstructure of the surface of each plastic plate. The adhesion, proliferation, and differentiation of the ESCs were compared and analyzed revealing differences in surface microstructure dependent on coating concentration. Results: According to AFM analysis, there was a clear difference in the microstructure of the surface according to the presence or absence of the gelatin coating, and it was confirmed that there was no difference at a concentration of 0.5% or more. ES cell also confirmed the difference in cell adhesion, proliferation, and differentiation according to the presence or absence of gelatin coating, and also it showed no difference over the concentration of 0.5%. Conclusions: The optimum gelatin-coating for the maintenance and differentiation of ES cells is 0.5%, and the gelatin concentration-mediated microenvironment and ES cell signaling are closely correlated.