PROJECT SUMMARY After injury, tendon function is often compromised due to poor healing and failure to regenerate native structure. Due to limited treatment options, there is an unmet clinical need for effective therapies that promote tendon healing and functional restoration. However, our incomplete understanding of tendon biology prevents the design of therapeutics that activate regenerative pathways involved in tendon formation. In particular, two major obstacles exist: (1) the critical regulators of tendon induction and differentiation are unknown and (2) the regulators driving non-regenerative adult tendon healing have not been elucidated or overcome. While the mouse is the gold standard model to study the biology of mammalian tendon development and healing, the extent of its relevance to human is unclear. To address these questions, we established robust differentiation protocols to derive tenocytes from mouse embryonic stem cells (mESCs) and human induced pluripotent stem cells (hiPSCs). Using these in vitro tendon differentiation models in combination with human and mouse embryos, clinically relevant injury models, and multiomics approaches, we will determine transcriptional and epigenetic regulation of tendon cell fate in the contexts of development and injury.