This work addresses the development of artificial cellular niches for studying the mechanisms that affect stem cell pluripotency, and aims at contributing to a better understanding of the cellular and molecular events that regulate stem cell functions. Embryonic stem (ES) cells were used as model systems to establish in vitro artificial microenvironments that better mimic the extracellular matrix. Microscale-based systems were designed for 3D culture of these cell types, and high-throughput screening platforms were developed to unveil the effects of complex interactions on cell fate. Particularly, We have developed a novel 3D cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. Our results revealed that this platform is suitable for studying the expansion of mouse ES cells as they retain their pluripotent and undifferentiated state. In addition to the influence of mechanical and matrix-related responses, the effects of microenvironmental conditions (e.g. small molecules and growth factors) were also analyzed due to their capacity of modulating intracellular pathways. This novel platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses.