Muscle stem cells (MuSCs) play a crucial role in maintaining muscle balance and repairing injuries in adult mammalian skeletal muscle. Located between the myofiber sarcolemma and a surrounding layer of extracellular matrix called the basal lamina, MuSCs are maintained in an inactive, resting state through a precisely regulated cellular environment comprising of both biochemical and physical cues. When injury occurs, cytokines released by inflammatory cells as well as a disruption of juxtacrine interactions prompt MuSCs to transition into an actively dividing state. External and intrinsic cues regulate a portion of activated MuSCs to commit along the myogenic differentiation axis and fuse into multinucleated myofibers. The exceptional regenerative ability of MuSCs positions them as a promising option for a new class of stem cell-based therapies aimed at treating impairments in muscle regeneration. However, these clinical efforts are bottlenecked by a limited understanding of the intricate interplay between MuSCs and their in vivo environment. As such, there exists an urgent need to study the external regulatory systems that influence and coordinate MuSC behavior. In this dissertation, I make use of biologically-inspired microenvironments to study the Notch signaling pathway, which occurs between MuSCs and neighboring cells. I demonstrate that Notch ligands vary in the strength of their influence on MuSC phenotype. I identify a drift in MuSC identity associated with extended culture on tissue plastic that correlates with a lowered reactivity with the Notch ligand Jag2. I connect this lack of reactivity with Jag2 to a specific interaction of Jag2 with Notch3. In this work, I also describe a novel method for asymmetric presentation of niche ligands to cultured cells using hydrogels tunable to biologically-relevant mechanical properties. The collection of this work represents a promising step forward towards in vitro control over MuSC self-renewal and subsequent development of autologous MuSC therapies.