The connections between energy metabolism and stemness of hematopoietic stem cells (HSCs) at different developmental stages remain largely unknown. We herein generate a transgenic mouse line for the genetically encoded NADH/NAD+ sensor (SoNar) and demonstrate that there exist three distinct fetal liver hematopoietic cell populations according to the ratios of SoNar fluorescence. SoNar-low cells have an enhanced level of mitochondrial respiration, but similar glycolytic level to SoNar-high cells. Interestingly, 10% of SoNar-low cells are enriched for 65% of total immunophenotypical fetal liver HSCs (FL-HSCs) and contain approximately 5-fold greater functional HSCs than that of SoNar-high counterparts. SoNar can monitor sensitively the dynamic changes of energy metabolism in HSCs both in vitro and in vivo. Mechanistically, STAT3 transactivates MDH1 to sustain the malate-aspartate NADH shuttle activity and the HSC self-renewal and differentiation. We reveal an unexpected metabolic program of FL-HSCs and provide a powerful genetic tool for metabolic studies of HSCs or other types of stem cells.