The vast majority of cellular ATP is produced by the oxidative phosphorylation (OXPHOS) system, which comprises the four complexes of the electron transfer chain plus the ATP synthase. Complex I is the largest of the OXPHOS complexes, and mutation of the genes encoding either the subunits or assembly factors of Complex I can result in Complex I deficiency, which is the most common OXPHOS disorder. Mutations in the Complex I gene NDUFS4 lead to Leigh syndrome, which is the most frequent presentation of Complex I deficiency in children presenting with progressive encephalopathy shortly after birth. Symptoms include motor and intellectual retardation, often accompanied by dystonia, ataxia, and growth retardation, and most patients die by 3 years of age. To understand the origins of this disease, we have generated a series of mouse embryonic stem cell lines from blastocysts that were wild type, heterozygous, and homozygous for the deletion of the Ndufs4 gene. We have demonstrated their pluripotency and potential to differentiate into all cell types of the body. Although the loss of Ndufs4 did not affect the stability of the mitochondrial and nuclear genomes, there were significant differences in patterns of chromosomal gene expression following both spontaneous differentiation and directed neural differentiation into astrocytes. The defect also affected the potential of the cells to generate beating embryoid bodies. These outcomes demonstrate that defects associated with Complex I deficiency affect early gene expression patterns, which escalate during early and later stages of differentiation and are mediated by the defect and not other chromosomal or mitochondrial DNA defects.