Stem cell fate can be influenced by metabolite levels in culture but it is unknown whether physiological variations in metabolite levels regulate stem cell function within normal tissues. We developed a metabolomics method for analysis of rare cell populations isolated directly from tissues and used it to compare haematopoietic stem cells (HSCs) to restricted haematopoietic progenitors. Each haematopoietic cell type had a distinct metabolic signature. Human and mouse HSCs had unusually high levels of ascorbate, which declined with differentiation. Systemic or cell autonomous ascorbate depletion in mice increased HSC frequency and function and promoted myelopoiesis. Ascorbate regulated HSC function in part through Tet2, a dioxygenase tumor suppressor enzyme. Ascorbate depletion cooperated with Flt3ITD leukaemic mutations to accelerate leukaemogenesis, and this was reversed by dietary ascorbate. Therefore physiological variations in ascorbate levels in vivo regulate Tet activity, regeneration and leukaemogenesis.