Immunotherapy has shifted the landscape of cancer treatment with the advent of checkpoint inhibitors, which have become the standard of care for multiple types of cancer. Despite the remarkable success of anti-PD1 and anti-CTLA-4, patient response rates to checkpoint blockade are often only 20-30%, necessitating the need for new therapeutic targets. Vdomain Ig Suppressor of T cell activation (VISTA) is an orthogonal checkpoint molecule that dampens the interaction between antigen-presenting cells and T cells to prevent effective anti-tumor responses. In this thesis, I present multiple projects related to the characterization of VISTA as a therapeutic target. First, I report the high-resolution crystal structure of the VISTA extracellular-domain and elucidate an important functional epitope of a clinical VISTA antibody (Chapter 2). I used a genetic and enzymatic de-glycoyslation approach to facilitate crystallization and utilized a display-based sorting strategy to isolate hotspot contact residues. Second, I detail the engineering and characterization of a unique, high affinity, species cross-reactive antibody against VISTA (Chapter 4). Using a combination of immune cell assays, epitope mapping strategies, and syngeneic mouse models, I demonstrate the efficacy of this antibody as a VISTA inhibitor. These efforts highlight a promising candidate for therapeutic development and provide a blueprint for future drug discovery campaigns against this promising new checkpoint target.