Summary Tissue homeostasis and regeneration are sustained by continuous self-renewal and differentiation of stem cells. Failure to balance these processes is associated with diverse and common public health issues, such as cancer and degenerative diseases. Discovering the mechanisms that regulate stem cell decisions is fundamentally important but has been stymied by the inability to study these dynamic processes in an intact mammal. My group overcomes this obstacle by leveraging the unique accessibility of the skin and our novel intravital imaging approaches to track epithelial cells in real time in live animals. We have so far used this approach to comprehensively identify all fate decisions within the epidermis, allowing us to uncover complex relationships between stem cell behaviors. This has led to our conceptually innovative model that stem cells are on a continuum of differentiation, coordinating their progression through the continuum based on tissue demand. Despite these advances, how cell fate decisions are molecularly orchestrated on the tissue-level remains unclear. Here, we adapt our intravital imaging techniques to visualize key molecular events – changes to transcription, chromatin structure, and signaling pathway activation – and determine their role in population-level coordination of cell fate decisions. We hypothesize that a global mechanism underlies the local coordination of stem cell states, and that this mechanism is essential to ensure skin homeostasis. To test this hypothesis, we will determine how differentiation is initiated (Aim 1). We identified a population of cells in the basal layer that co- express stem- and differentiation-markers but do not display morphological signs of differentiation. To determine if these cells are in a reversible transition from stemness to differentiation, we will interrogate their transcriptional plasticity, chromatin rearrangements, and cell-extrinsic regulators. In Aim 2, we will define intercellular signaling patterns that coordinate stem cell and differentiation behaviors. Calcium signaling acts as a signal integrator in the late stages of epidermal differentiation and in wound-healing, and our preliminary data show that calcium signaling is prevalent and dynamic in the basal epidermis. To define how calcium signaling intersects with the coordination of stem cell fate decisions, we will uncover the high-order dynamics of calcium signaling in the basal layer and determine how calcium signaling regulates the coordination of cell cycles, self-renewal, and differentiation of basal cells. To achieve these aims, we will use an integrated approach of cutting-edge imaging technology, transcriptomics, mouse genetics, and machine learning. This research is significant because we expect to uncover global molecular mechanisms that coordinate stem cell self-renewal and differentiation in a live mammal. Our findings will likely drive innovation in related fields, given that many principles of stem...