Stem cell therapy and tissue engineering offer great potential for the treatment of a variety of musculoskeletal disorders . However, the study of stem cells is challenged to identify the most robust cells and control the fate of those cells. Live cell imaging is a tool that offers a high-throughput approach to studying a number of cell types and conditions to help elucidate cells and reagents, which are most functional for treating injuries of bone, cartilage, muscle, or tendon/ligaments. In vitro time-lapsed microscopic imaging of living cells is an efficient approach to generating large datasets on dynamic stem cell behavior. This report describes the live cell imaging tools and their use to observe and track unique stem cell activities. The novel aspect of these systems include automation of imaging by robotic movement of cell culture flasks, which enables selection of a large number of regions of interest for data collection. Standard to most such systems is an environmentally controlled chamber to maintain experimental conditions, including temperature, gas levels, and humidity, so that stem cells can be tracked by visible and epifluorescence imaging over extended periods. These systems offer the capability to overcome limitations associated with scarcity of stem cells, or frequency of events, such as myotube contraction. They can be used to examine fluxes in mitochondrial membrane potential , or create dynamic coculture environments where individual subpopulations can be identified. In this report, we provide an example of the system's use to identify donor cell variability using human umbilical cord mesenchymal stem cells . We describe automation in time-lapsed microscopic imaging and how this technology is providing new insights into stem cell biology .