Cell interactions with the extracellular matrix (ECM) play a critical role in their physiology. Here, we sought to determine the role of exogenous and endogenous ECM in the differentiation of nonhuman primate ESCs. We evaluated cell differentiation from expression of lineage gene mRNA and proteins using real-time polymerase chain reaction and immunohistochemistry. We found that ESCs that attached to and spread upon highly adhesive collagen do not differentiate efficiently, whereas on the less adhesive Matrigel, ESCs form aggregates and differentiate along mesoderm and especially endoderm lineages. To further decrease ESC attachment to the substrate, we cultured them either on nonadhesive agarose or in suspension. In both cases, ESCs formed aggregates and efficiently differentiated along endoderm and mesoderm lineages, most strikingly into cardiomyocytes. Aggregates formed by thus-differentiated ESCs started to beat with a frequency of 50–100 beats per minute and continued to beat for approximately a month. In spite of the presence of exogenous ECM, ESCs were dependent on endogenous ECM for their survival and differentiation, as the inhibition of endogenous collagen induced a gradual loss of ESCs and neither a simple matrix, such as type I collagen, nor the complex matrix Matrigel was able to rescue these cells. In conclusion, adhesiveness to various ECM and nonbiological substrates determines the differentiation of ESCs in such a way that efficient cell-cell aggregation, together with less efficient cell attachment and spreading, results in more efficient cell differentiation.