Testis development and function is regulated by intricate cell-cell cross talk. Characterization of the mechanisms underpinning this has been derived through a wide variety of approaches including pharmacological manipulation, transgenics, and cell-specific ablation of populations. The removal of all or a proportion of a specific cell type has been achieved through a variety of approaches. In this paper, we detail a combined transgenic and pharmacological approach to ablate the Sertoli or germ cell populations using diphtheria toxin in mice. We describe the key steps in generation, validation, and use of the models and also describe the caveats and cautions necessary. We also provide a detailed description of the methodology applied to characterize testis development and function in models of postnatal Sertoli or germ cell ablation.