Type 1 diabetes (T1D) represents a hallmark of the fatal multiorgan autoimmune syndrome affecting humans with abrogated Foxp3 regulatory T (Treg) cell function due to gene mutations, but whether the loss of Foxp3 Treg cell activity is indeed sufficient to promote β cell autoimmunity requires further scrutiny. As opposed to human Treg cell deficiency, β cell autoimmunity has not been observed in non-autoimmune-prone mice with constitutive deficiency or after diphtheria toxin receptor (DTR)-mediated ablation of Foxp3 Treg cells. In the spontaneous nonobese diabetic (NOD) mouse model of T1D, constitutive Foxp3 deficiency did not result in invasive insulitis and hyperglycemia, and previous studies on Foxp3 Treg cell ablation focused on Foxp3 NOD mice, in which expression of a transgenic BDC2.5 T cell receptor (TCR) restricted the CD4 TCR repertoire to a single diabetogenic specificity. Here we revisited the effect of acute Foxp3 Treg cell ablation on β cell autoimmunity in NOD mice in the context of a polyclonal TCR repertoire. For this, we took advantage of the well-established DTR/GFP transgene of DEREG mice, which allows for specific ablation of Foxp3 Treg cells without promoting catastrophic autoimmune diseases. We show that the transient loss of Foxp3 Treg cells in prediabetic NOD.DEREG mice is sufficient to precipitate severe insulitis and persistent hyperglycemia within 5 days after DT administration. Importantly, DT-treated NOD.DEREG mice preserved many clinical features of spontaneous diabetes progression in the NOD model, including a prominent role of diabetogenic CD8 T cells in terminal β cell destruction. Despite the severity of destructive β cell autoimmunity, anti-CD3 mAb therapy of DT-treated mice interfered with the progression to overt diabetes, indicating that the novel NOD.DEREG model can be exploited for preclinical studies on T1D under experimental conditions of synchronized, advanced β cell autoimmunity. Overall, our studies highlight the continuous requirement of Foxp3 Treg cell activity for the control of genetically pre-installed autoimmune diabetes.