We present development and use of a 3D synthetic extracellular matrix (ECM) analog with integrin-specific adhesion ligands to characterize the microenvironmental influences in embryonic stem cell (ESC) self-renewal. Transcriptional analysis of 24 integrin subunits followed by confirmation at the translational and functional levels suggested that integrins alpha(5)beta(1), alpha(v)beta(5), alpha(6)beta(1) and alpha(9)beta(1) play important roles in maintenance of stemness in undifferentiated mouse ESCs. Using the well-defined matrix as a tool to activate integrins alpha(5)beta(1) plus alpha(v)beta(5), alpha(6)beta(1) and alpha(9)beta(1), individually and in combination, differential integrin activation was demonstrated to exert exquisite control over ESC fate decisions. Simultaneous ligation of these four integrin heterodimers promoted self-renewal, as evidence by prolonged SSEA-1, Oct4 and Nanog expression, and induced Akt1 kinase signaling along with translational regulation of other stemness-related genes. The biofunctional network we have designed based on this knowledge may be useful as a defined niche for regulating ESC pluripotency through selective cell-matrix interactions, and the method we present may be more generally useful for probing matrix interactions in stem cell self-renewal and differentiation.