Blood formation is coordinated by a set of functionally heterogeneous haematopoietic stem cell (HSC) compartments both in mouse and in human. However, how this diversity is regulated at the cellular and molecular level and when HSC multipotency is lost is still not known, in particular in humans. Quiescence is an important property of HSCs and fine regulation of the balance between quiescence and cell cycle entry is of vital importance for maintaining a healthy HSC pool and avoid haematological malignancies. Very little is known about how molecular networks change during exit from quiescence and no studies have formally examined if cell fate decisions occur during this process or later on during the cell cycle. Here, I combine index sorting, in-vitro single cell functional assays and single cell RNA-sequencing with xenotransplantation assays to profile single human HSC properties in the purest HSC compartment reported to date (CD49+ HSCs) and to understand when the first steps of lineage restriction occur during HSC differentiation. Moreover, I use an in-vitro model system to comprehensively study HSC activation and to investigate whether HSC self-renewal is lost during quiescence exit or during cell cycle progression. First, I unveil an unexpected degree of intrinsic functional and molecular heterogeneity within the human CD49f+ HSC pool. I demonstrate that the first restriction step towards the lymphoid lineage occurs already within the CD49f+ HSC compartment and generates erythroid-deficient myeloid-lymphoid committed cells. Within this compartment, transcriptional programmes and lineage potential progressively change along a gradient of opposing cell surface expression of CLEC9A and CD34. Two functionally distinct populations can be identified and purified. CLEC9Ahi CD34lo cells contain long-term repopulating multipotent HSCs with slow quiescence exit kinetics, whereas CLEC9Alo CD34hi cells are restricted to myelo-lymphoid differentiation and display infrequent but durable repopulation capacity. Second, I demonstrate that a drastic transcriptional remodelling reflective of the fast metabolic activation seen in ex-vivo cultured HSCs occurs during quiescence exit, and independently of cell cycle progression. In-vivo data show that the reduction in repopulation capacity that accompanies HSC culture also occurs independently of cell cycle progression. Recent work highlighted the active role of mitochondria and metabolism in HSC fate decision and self-renewal. My data is consistent with a model in which the metabolic remodelling seen during exit from quiescence represents the first step towards loss of self-renewal and differentiation. This has important implications for improving ex-vivo protocols for HSC culture and HSC transplants.