Inhibitory checkpoint blockade has significantly improved patient response rate across numerous tumor types. However, most patients remain unresponsive to immunotherapy, suggesting that unappreciated mechanisms of resistance exist. The tumor microenvironment (TME) is unique and composed of many suppressive cell populations that inhibit antitumor immune responses, including regulatory T cells (Tregs). The TME is nutrient poor, acidic, and hypoxic, creating a challenging microenvironment for immune cells to function and survive. Tregs suppress a wide variety of cell populations through multiple mechanisms and are tasked with limiting tissue damage. Tregs are now considered to be a barrier to effective antitumor immunity. Systemic Treg depletion is not favored because of their critical role in maintaining immune homeostasis and preventing autoimmunity. Reducing Treg function specifically within the TME may provide a more effective, targeted approach to limit the immunosuppressive environment within the tumor without inducing systemic adverse consequences. Targeting molecules that cause Treg instability, characterized by loss of critical Treg transcription factors such as Foxp3, could result in conversion into cells that cause immune pathology, tissue damage, and subsequent autoimmune side effects. Interferon-γ (IFNγ) can cause intratumoral Treg “fragility,” which results in loss of suppressive activity and increased IFNγ production without loss of Foxp3 expression and gross Treg “identity.” We reviewed the impact Tregs have on the TME and vice versa, and their implications for responsiveness to cancer immunotherapy. We propose that the extent to which intratumoral Tregs develop a “fragile” phenotype following immunotherapy will predict and dictate responsiveness. Cancer Immunol Res; 6(8); 882–7. ©2018 AACR.