Hematopoietic stem cells (HSCs) are responsible for life-long production of all mature blood cells. This unique characteristic makes them an ideal candidate for cell-based therapies to treat various hematological malignancies. Their extensive use in the clinic is often hampered due to insufficient number of cells obtained from donors. Countless attempts to expand HSCs in vitro have failed, primarily due to our inability to recapitulate key features of the native bone marrow microenvironment, termed niche, in a dish. The absence of important niche signals in vitro results in rapid proliferation of HSCs with a concomitant loss of their long-term multi lineage blood reconstitution potential. The niche in the bone marrow involves a highly complex network of physical and biochemical signals that, in concert with cell-intrinsic mechanisms, is believed to control HSC fate choices. Moreover, the hypoxic conditions in the niche presents an extreme metabolic environment, imposing HSCs to attain a distinct metabolic identity as compared to their differentiated progeny. However, despite decades of research it is currently very poorly understood how HSCs take the decision to either undergo self-renewal or differentiation. Insights into the mechanisms regulating HSC fate choices are key to design better strategies for HSC maintenance and expansion in vitro for use in clinical transplantations. The overall goal of this thesis is to employ innovative experimental tools to explore the role of metabolism in regulating HSC fate choices. In the first part of this thesis a versatile cell-tracking assay was developed to follow HSC divisions in vitro. A combination of cell tracking and immunostaining was used to systematically map phenotypic changes in HSCs up to four divisions, under defined culture conditions imposing specific fates. We found that the proportion of cells maintaining an HSC phenotype decreased with increasing number of cell divisions, supporting the notion that faster cycling results in HSC exhaustion. In the second part of this thesis, we for the first time identify a link between mitochondrial metabolism and HSC fate decision. Using flow cytometry and long-term blood reconstitution assays low mitochondrial activity was established as a reliable marker of functional HSCs, independent of their cell cycle state. Consequently, we could use this marker to reliable identify self-renewing HSCs from heterogeneous in vitro cultures. Strikingly, we found that HSC fate could be altered by artificially modulating their mitochondrial activity in vitro. These results suggest that mitochondrial activity is a determinant of HSC fate. The last part of this thesis describes an experimental paradigm to analyze in vivo niche-instructed fate choices in paired HSC daughter cells. Live single cell imaging revealed a significant increase in asynchronous divisions in niche activated HSCs compared to control cells, suggesting a possible involvement of niche-instructed asymmetric cell division program. Indeed, a significantly higher level of asymmetric gene expression was found in paired daughter cells arising from niche-instructed HSCs. This analysis led to the identification of 12 asymmetrically expressed genes, among them were key enzymes belonging to glycolytic and mitochondrial TCA cycle metabolic pathways. Altogether, this thesis successfully employed unique experimental strategies to provide an intriguing link between metabolism and HSC fate choices.