Embryonic stem (ES) cells are one of the principle focuses in biology because of their pluripotency and potential therapeutic applications. Although it is known that soluble factors affect differentiation of ES cells, little is known about the influence of mechanical stimulus on the differentiation of ES cells. Here we investigate the influence of mechanical stimuli on mRNA expression of mouse ES cell during the differentiation process. Without a mechanical stimulus, mRNA levels of pluripotency markers, Oct3/4 and Klf4 decreased and primitive ectoderm marker, Lefty1 mRNA level increased, suggesting that mouse ES cells might differentiate into primitive ectoderm. With a mechanical stimulus, Klf4 and Lefty1 mRNA levels decreased. We speculate that progress of differentiation into primitive ectoderm can be inhibited by a mechanical stimulus.