Stem cells maintain tissue homeostasis by driving cellular turnover and regeneration upon damage. They reside within specialized niches that provide the signals required for stem cell maintenance. Stem cells have been identified in many tissues and cancer types, but their behavior within the niche and their reaction to microenvironmental signals were inferred from limited static observations. Recent advances in live imaging techniques, such as live cell imaging and intravital microscopy, have allowed the visualization of stem cell behavior and dynamics over time in their (near) native environment. Through these recent technological advances, it is now evident that stem cells are much more dynamic than previously antici-pated, resulting in a model in which stemness is a state that can be gained or lost over time. In this review, we will highlight how live imaging and intravital microscopy have unraveled previously unanticipated stem cell dynamics and plasticity during development, homeosta-sis, regeneration, and tumor formation.