Mechanistic and functional studies by gene disruption or editing approaches often suffer from confounding effects like compensatory cellular adaptations generated by clonal selection. These issues become particularly relevant when studying factors directly involved in genetic or epigenetic maintenance. To provide a genetic tool for functional and mechanistic investigation of DNA repair mediated active DNA demethylation, we generated a Tdg minigene model in mice and mouse embryonic stem cells (ESCs). The floxed miniTdg is rapidly and reliably excised by tamoxifen-induced Cre expression in mice and ESCs, depleting TDG to undetectable levels within 24 hours of induction. We describe the functionality of the engineered miniTdg in mouse and ESCs (TDGiKO ESCs) and validate the pluripotency and differentiation potential of TDGiKO ESCs as well as the phenotype of induced TDG depletion. The controlled and rapid depletion of TDG allows for a precise manipulation at any time point in multistep experimental procedures as presented here for neuronal differentiation in vitro. Thus, we provide a thoroughly validate d genetic tool for the functional and mechanistic investigation of TDG in active DNA (de)methylation and/or DNA repair with minimal interference from adaptive effects and clonal selection.