The α-cells of the pancreatic islets are defined by their hormone, glucagon, whichthey release in response to signals indicating increased demand for glucose in the body. They exist in dynamic co-regulation with their neighboring β-cells, which release insulin to lower blood glucose, and δ-cells, which secrete somatostatin to impose local inhibitory feedback in the islet. The activity of all three cells are largely reliant on circulating glucose levels, but α-cells are also activated by nutrients like amino acids and hormones like epinephrine and arginine vasopressin. This system is disrupted in diabetes, and glucagon dysfunction ultimately contributes greatly to diabetic hyperglycemia and impaired counterregulation. Understanding what goes wrong in diabetes is complicated by that fact that while many physiological signals are known to regulate glucagon secretion, α-cells are often viewed as a single population and thus individual α-cell responses are not well characterized. In this dissertation, we have applied improved genetic tools for expressing fluorescent biosensors specifically in mouse α-cells in order to gain a population-level view of their activity in intact islets in real time. In doing so we are able to interrogate how intra-islet signaling and various glucagon secretagogues, as well as diabetes, shape α-cell behavior. Chapter 1 provides a comprehensive overview of islet biology, including the changes that occur in diabetes. Chapter 2 investigates the role of another β-cell hormone, Urocortin3, in inhibitory paracrine feedback on α-cells. Chapter 3 characterizes functional heterogeneity in α-cell activation by common physiological stimuli and how diabetes affects these responses. Chapter 4 provides a summary of the work performed, concluding remarks, and plans for future studies.