Extracellular matrix (ECM) components regulate stem-cell behavior, although the exact effects elicited in embryonic stem (ES) cells are poorly understood. We previously developed a simple, defined, serum-free culture medium that contains leukemia inhibitory factor (LIF) for propagating pluripotent mouse embryonic stem (mES) cells in the absence of feeder cells. In this study, we determined the effects of ECM components as culture substrata on mES cell self-renewal in this culture medium, comparing conventional culture conditions that contain serum and LIF with gelatin as a culture substratum. mES cells remained undifferentiated when cultured on type I and type IV collagen or poly-D-lysine. However, they differentiated when cultured on laminin or fibronectin as indicated by altered morphologies, the activity of alkaline phosphatase decreased, Fgf5 expression increased, and Nanog and stage-specific embryonic antigen 1 expression decreased. Under these conditions, the activity of signal transducer and activator of transcription (STAT)3 and Akt/protein kinase B (PKB), which maintain cell self-renewal, decreased. In contrast, the extracellular signal-regulated kinase (ERK)1/2 activity, which negatively controls cell self-renewal, increased. In the defined conditions, mES cells did not express collagen-binding integrin subunits, but they expressed laminin- and fibronectin-binding integrin subunits. The expression of some collagen-binding integrin subunits was downregulated in an LIF concentration-dependent manner. Blocking the interactions between ECM and integrins inhibited this differentiation. Conversely, the stimulation of ECM-integrin interactions by overexpressing collagen-binding integrin subunits induced differentiation of mES cells cultured on type I collagen. The results of the study indicated that inactivation of the integrin signaling is crucial in promoting mouse embryonic stem cell self-renewal. Disclosure of potential conflicts of interest is found at the end of this article.