The concept of using stem cells as self-renewing sources of healthy cells in regenerative medicine has existed for decades, but most applications have yet to achieve clinical success. A main reason for the lack of successful stem cell therapies is the difficulty in fully recreating the maintenance and control of the native stem cell niche. Improving the performance of transplanted stem cells therefore requires a better understanding of the cellular mechanisms guiding stem cell behavior in both native and engineered three-dimensional (3D) microenvironments. Most techniques, however, for uncovering mechanisms controlling cell behavior in vitro have been developed using 2D cell cultures and are of limited use in 3D environments such as engineered tissue constructs. Deciphering the mechanisms controlling stem cell fate in native and engineered 3D environments, therefore, requires rigorous quantitative techniques that permit mechanistic, hypothesis-driven studies of cell-microenvironment interactions. Here, we review the current understanding of 2D and 3D stem cell control mechanisms and propose an approach to uncovering the mechanisms that govern stem cell behavior in 3D.