Muscle stem cells (MuSCs) are the cells mainly responsible for the regenerative capacity of skeletal muscle. MuSCs reside in a stem cell “niche” microenvironment which directs their fate and function, largely through interactions with surrounding niche factors and cells. Notably, muscle capillary endothelial cells (ECs) are near MuSCs in homeostasis and are essential for muscle regeneration, but specific signaling interactions between MuSCs and ECs remain unclear. We employed a single-cell RNA sequencing atlas of skeletal muscle regeneration and a ligand-receptor model to identify Dll4-Notch3, Hbegf-Cd82, and Cxcl12-Sdc4 as new candidate cell-cell interactions between capillary EC ligands and MuSC receptors in both homeostasis and regeneration. Through in vitro experiments, we found that MuSCs co-cultured in direct contact with skeletal muscle-derived ECs (SkMECs) significantly increased MuSC proliferation, whereas direct co-culture with a non-skeletal muscle EC line and culture with EC conditioned media did not affect MuSC proliferation. We used bulk RNA-sequencing to compare cultured ECs from skeletal muscle and non-skeletal muscle sources with in vivo skeletal muscle ECs isolated at various time points post-injury to investigate the maintenance of a tissue-specific gene signature in vitro. These results revealed significant differences in gene expression between in vivo and cultured ECs, and found SkMECs best preserved a skeletal muscle-specific endothelial signature. Co-culture experiments with SkMECs and muscle progenitor cells (MPCs) found that co-culture increased the total number of MPCs and the percentage of Pax7+ MPCs compared to monoculture, suggesting that EC-derived factors increase MPC self-renewal. We found that Notch inhibition with co-culture dramatically decreased MPC proliferation and increased myogenic differentiation. Others have reported that EC-MuSC Notch signaling induces MuSC quiescence. Our results suggest that Notch signaling between ECs and MuSCs may influence MuSCs during an activated state, potentially affecting regeneration and MuSC self-renewal. Overall, this work underscores the significant role of endothelial-derived signaling in MuSC function and provides a comprehensive evaluation of EC-sourcing for establishing a skeletal muscle-specific co-culture platform, paving the way for future studies on EC-MuSC ligand-receptor interactions.