Regeneration is the regrowth of damaged tissues and structures, which encompasses a complex process of cell activation and coordination. Freshwater flatworms called planarians are incredibly regeneration-competent, with the ability to regenerate virtually all body parts. Therefore, understanding the fundamental cellular mechanisms that promote planarian regeneration can unveil more universal themes required for this heightened level of healing in animals. While several essential planarian traits and genetic responses for regeneration are identified, less is known about the mechanisms that drive and coordinate these processes after injury. In this dissertation, I take a dual approach – investigating both upstream signaling ligands and downstream signal transducers – to define potential signaling pathways necessary for successful regeneration in the planarian species Schmidtea mediterranea. Regarding the upstream signals, by targeting a key neuropeptide processing enzyme-encoding gene, prohormone convertase 2 (pc2), I show that putative neuropeptide signaling plays many important roles including promoting proper animal behavior, robust regeneration, and tissue maintenance. Additionally, I uncovered a novel role for planarian pc2 in stem cell differentiation, which I propose is the primary phenotype resulting in defects to regeneration and tissue maintenance. Regarding the downstream signal transducers, I explored potential roles for G protein-coupled receptor pathways in planarian regeneration via the canonical downstream heterotrimeric G proteins. I identified and phylogenetically classified all the heterotrimeric G protein subunit genes in S. mediterranea, then performed a functional characterization for these subunits. I found that the planarian heterotrimeric G protein subunits showed diverse roles including promoting proper behaviors, survival, and regeneration. I further show that two subunits critical for regeneration, Gαq1 and Gβ1-4a, facilitate reestablishment of key polarity domains, which is a primary outcome contributing to regeneration in these animals. My results support the hypothesis that neuropeptide- and GPCR-related signaling pathways play crucial roles in stem cell regulation and planarian regeneration. This work provides a foundation for future investigation into these important gene families as well as specific candidate pathways to continue exploring. Further delineating these pathways will provide valuable knowledge to how an animal regulates pluripotent stem cells in vivo and coordinates cellular responses to achieve complex tissue regeneration.