A single hematopoietic stem cell (HSC) is capable of reconstituting hematopoiesis and maintaining homeostasis by balancing self-renewal and cell differentiation. The mechanisms ofHSCdivision balance, however, are not yet defined. Herewe demonstrate, by characterizing at the single-cell level a purified and minimally heterogeneous murine Tie2(+) HSC population, that these top hierarchical HSCs preferentially undergo symmetric divisions. The induction ofmitophagy, a quality control process in mitochondria, plays an essential role in self-renewing expansion of Tie2(+) HSCs. Activation of the PPAR (peroxisome proliferator-activated receptor)-fatty acid oxidation pathway promotes expansion of Tie2(+) HSCs through enhanced Parkin recruitment in mitochondria. These metabolic pathways are conserved in human TIE2(+) HSCs. Our data thus identify mitophagy as a key mechanism of HSC expansion and suggest potential methods of cell-fate manipulation through metabolic pathways.