Mitochondrial abundance and genome are crucial for cellular function, with disruptions often associated with disease. However, methods to modulate these parameters for direct functional dissection remain limited. Here, we eliminate mitochondria from pluripotent stem cells (PSCs) by enforced mitophagy and show that PSCs survived for several days in culture without mitochondria. We then leverage enforced mitophagy to generate interspecies PSC fusions that harbor either human or non-human hominid (NHH) mitochondrial DNA (mtDNA). Comparative analyses indicate that human and NHH mtDNA are largely interchangeable in supporting pluripotency in these PSC fusions. However, species divergence between nuclear and mtDNA leads to subtle species-specific transcriptional and metabolic variations. By developing a transgenic enforced mitophagy approach, we further show that reducing mitochondrial abundance leads to delayed development in pre-implantation mouse embryos. Our study opens avenues for investigating the roles of mitochondria in development, disease, and interspecies biology.