Tissue stem cells and germ line or embryonic stem cells were shown to have reduced oxidative metabolism, which was proposed to be an adaptive mechanism to reduce damage accumulation caused by reactive oxygen species. However, an alternate explanation is that stem cells are less dependent on specialized cytoplasmic functions compared with differentiated cells, therefore, having a high nuclear-to-cytoplasmic volume ratio and consequently a low mitochondrial content. To determine whether stem cells rely or not on mitochondrial respiration, we selectively ablated the electron transport chain in the basal layer of the epidermis, which includes the epidermal progenitor/stem cells (EPSCs). This was achieved using a loxP-flanked mitochondrial transcription factor A (Tfam) allele in conjunction with a keratin 14 Cre transgene. The epidermis of these animals (Tfam(EKO)) showed a profound depletion of mitochondrial DNA and complete absence of respiratory chain complexes. However, despite a short lifespan due to malnutrition, epidermal development and skin barrier function were not impaired. Differentiation of epidermal layers was normal and no proliferation defect or major increase of apoptosis could be observed. In contrast, mice with an epidermal ablation of prohibitin-2, a scaffold protein in the inner mitochondrial membrane, displayed a dramatic phenotype observable already in utero, with severely impaired skin architecture and barrier function, ultimately causing death from dehydration shortly after birth. In conclusion, we here provide unequivocal evidence that EPSCs, and probably tissue stem cells in general, are independent of the mitochondrial respiratory chain, but still require a functional dynamic mitochondrial compartment.