Murine embryonic stem cells (ESCm) cultured in vitro in the presence of LIF (leukemia inhibitory factor) maintain pluripotency. However, when LIF is removed from the media, an active differentiation into various specialized somatic cells is observed. The aim of the study was to determine the role of substrate stiffness in maintaining of pluripotency of embryonic stem cells in vitro culture. To this aim, we used the method of culturing pluripotent stem cells in vitro, the method of “hanging drop”, the determination of the Young’s modulus for polyacrylamide gel of different hardness, the immunocytochemical alkaline phosphatase (AP) streptavidin-biotin method, microscopy. By culturing ESCm on a soft, medium and hard density polyacrylamide gel as a substrate (0.8, 4.0, 8.0 кPа), we found that on a soft gel ESCm differentiation does not occur even in the absence of LIF. ESCm cultured on a soft substrate continue to show signs of pluripotency, namely, create round compact colonies with high alkaline phosphatase activity and form embryoid bodies (EB), the efficiency of which (87.5 ± 3.2 per 100 cells seeded) did not decrease even after LIF withdrawal. In the absence of LIF, ESCs cultured on a hard base showed a low level of EB formation (23.5 ± 2.24). The results of our observations demonstrate that the process of EB formation may be influenced not only by a composition of nutrient medium, but also by complex interaction between the physical forces of the matrix and the mechanical properties of 3D cell aggregates. The model is considered as a tool to study early events in embryogenesis in the search of conditions for effective culture of progenitor cells and differentiated cells for transplantation.