ABSTRACT The majority of research on muscle development focuses on the specific regulation and direct contribution of myogenic progenitors and myoblasts to muscle formation. Recently, non-myogenic mesenchymal cells (NMMCs) located within developing muscle tissues have been identified as critical regulators of normal myogenesis. These studies show that muscle development or regeneration cannot occur in the absence of specific non-myogenic mesenchymal “niche” cells. In order to genetically examine regulatory mechanisms that may be critical for NMMC function and normal muscle development, our lab has demonstrated the utility of the Prx1Cre in accessing the broad class of NMMCs within skeletal muscle. Flow cytometry and scRNA sequencing of the Prx1 lineage not only confirmed that multiple populations of NMMCs exist within the skeletal muscle, but also that these populations are responsive to NOTCH signaling. Further, preliminary results indicate that a conditional loss of NOTCH within NMMCs (NOTCH LOF, Prx1Cre; RBPJkf/f) impairs normal muscle development and function. Given this, we hypothesize that NOTCH mediated regulation of NMMCs is critical for normal skeletal muscle development and function. In testing this hypothesis, we will utilize a combination of in vivo and in vitro methods in order to: (Aim 1) determine the role of NOTCH signaling in non-myogenic mesenchymal progenitors during muscle development in vivo and (Aim 2) evaluate the cellular mechanisms by which NMMC-mediated NOTCH signaling regulates skeletal muscle in vitro. Our findings will further our fundamental understanding of muscle development, specifically, our understanding of niche regulation and dynamics during muscle formation and function. Additionally, these findings may provide key insights into the role(s) of NMMC-derived NOTCH signaling in diseases affecting muscle development, and may also aid in future studies of muscle regeneration where disruption of the NMMC niche prevents proper myogenesis.