High throughput screening (HTS) methodologies allow for simplified analysis of numerous conditions. The development of the arrayed cellular microenvironment technology has allowed for multi-factorial investigations into cellular processes through controlling the cellular environment. Here, we describe the development of a HTS cellular microarray technology and its application to (1) the identification and analysis of an optimal, defined matrix composition for human embryonic stem cells (hESCs), (2) the testing of synthetic polymers for their ability to support hESC growth, and (3) the development of a functional genomics screening platform. Through examination of gene expression of hESCs cultured on the defined matrix identified, we characterized maintenance of pluripotency and demonstrated differentiation potential. Additionally, polymers were systematically screened for ability to support hESC culture. Long-term culture methods on discovered polymers were examined through culture on hydrogels. Furthermore, we investigated a novel method of siRNA screening using this technology. The versatile arrayed cellular microenvironment technology can be utilized to understand the complex factors that modulate cellular activity and influence cell adherence, proliferation, and fate.