High throughput screens (HTS) of biological and synthetic molecules has become a useful tool in drug discovery and basic biology. However, traditional HTS can be cost- prohibitive given the costs of purified biological molecules and the rarity of certain cell types. Additionally, these approaches employ a candidate based strategy ignoring the complex crosstalk that occurs between combinations of biological molecules. To that end, we have developed a technology platform, called arrayed cellular microenvironments (ACME), which allows for the real-time simultaneous screening of thousands of biological and synthetic physiochemical parameters on cell attachment, proliferation, differentiation and gene expressions. We demonstrate that our platform provides data comparable to that obtained from traditional multi- well based assays while using 1,000-10,000 times fewer cells and reagents. As proof of principle, we applied this technology to identify combinations of microenvironment components that differentially modulate the phenotype of hepatic stellate cells (HSCs), a progenitor cell population of the liver that are involved in liver homeostasis. Next, we modified the ACME technology to screen the full complement of factors that may regulate human embryonic stem cell (hESC) proliferation and maintenance of pluripotency. Through the systematic screening of extracellular matrix proteins (ECMPs) and other signaling molecules, we developed and characterized a completely defined culture system for the long-term self -renewal of three independent hESC lines. Finally, we extended the ACME technology to screen synthetic polymers and peptides to identify artificial matrices that support self-renewal of hESCs. This system will be useful for future stem cell research, including the elucidation of differentiation protocols, as well as the identification of culture conditions of rare and recalcitrant primary cell populations, such as progenitor, adult stem, and cancer stem cells.