Regulatory T cells (Treg) have become an important player in regulating anticancer immune responses. In fact, published studies describe a correlation between tumor-infiltrating Tregs and poor prognosis. Once called "suppressor T cells," these T cells evaded isolation because of a lack of known markers that distinguished them from other T cells. However, the biology of these T cells is currently a major focus of immunologic research. Markers have since been discovered that identify these T cells and provide insights into how these T cells are regulated. Despite these advances, much needs to be learned about the subsets of Tregs and their specific roles in regulating immune responses. In addition, specific agents that target Tregs are currently unavailable. Cyclophosphamide has emerged as a clinically feasible agent that can suppress Tregs and allow more effective induction of antitumor immune responses. This review focuses on the use of cyclophosphamide in targeting Tregs to augment cancer vaccine approaches. However, these principles can also be applied to other immunotherapy strategies.