Cost-effective and scalable synthetic matrices that support long-term expansion of human pluripotent stem cells (hPSCs) have many applications, ranging from drug screening platforms to regenerative medicine. Here, we report the development of a hydrogel-based matrix containing synthetic heparin-mimicking moieties that supports the long-term expansion of hPSCs (≥20 passages) in a chemically defined medium. HPSCs expanded on this synthetic matrix maintained their characteristic morphology, colony forming ability, karyotypic stability, and differentiation potential. We also used the synthetic matrix as a platform to investigate the effects of various physicochemical properties of the extracellular environment on the adhesion, growth, and self-renewal of hPSCs. The observed cellular responses can be explained in terms of matrix interface-mediated binding of extracellular matrix proteins, growth factors, and other cell-secreted factors, which create an instructive microenvironment to support self-renewal of hPSCs. These synthetic matrices, which comprise of "off-the-shelf" components and are easy to synthesize, provide an ideal tool to elucidate the molecular mechanisms that control stem cell fate.