ABSTRACT: Maintenance of the blood system is dependent on dormant haematopoietic stem cells (HSCs) with long-term self-renewal capacity. Upon injury these cells are induced to proliferate in order to quickly re-establish homeostasis. The signalling molecules promoting the exit of HSCs out of the dormant stage remain largely unknown. Here we show that in response to treatment of mice with interferon-alpha (IFNα), HSCs efficiently exit G0 and enter an active cell cycle. HSCs respond to IFNα treatment by increased phosphorylation of STAT1 and PKB/Akt, expression of IFNα target genes and up-regulation of stem cell antigen-1 (Sca-1). HSCs lacking either the interferon-α/β receptor (IFNAR), STAT1 or Sca-1 are insensitive to IFNα stimulation, demonstrating that STAT1 and Sca-1 mediate IFNα induced HSC proliferation. Although dormant HSCs are resistant to the anti-proliferative chemotherapeutic agent 5-FU1, HSCs pre-treated (primed) with IFNα and thus induced to proliferate are efficiently eliminated by 5-FU exposure in vivo. Conversely, HSCs chronically activated by IFNα are functionally compromised and are rapidly out competed by non-activatable IFNAR-/- cells in competitive repopulation assays. In summary, while chronic activation of the IFNα pathway in HSCs impairs their function, acute IFNα treatment promotes the proliferation of dormant HSCs in vivo. These data may help to clarify the so far unexplained clinical effects of IFNα on leukemic cells and raise the possibility for novel applications of type I interferons to target cancer stem cells. cDNA microarray analysis was performed on sorted Lin neg, cKit+, CD150+, CD48neg HSCs from IFNα treated (16h after treatment) and untreated (littermate) mice. Per condition 3 independent biological replicates were analysed.