Haematopoietic stem cells (HSCs) are capable of shifting from a state of relative quiescence under homeostatic conditions to rapid proliferation under conditions of stress. The mechanisms that regulate the relative quiescence of stem cells and its association with self-renewal are unclear, as is the contribution of molecular regulators of the cell cycle to these decisions. Understanding the mechanisms that govern these transitions will provide important insights into cell-cycle regulation of HSCs and possible therapeutic approaches to expand HSCs. We have investigated the role of two negative regulators of the cell cycle, p27^Kip1 and MAD1, in controlling this transition. Here we show that Mad1^−/−p27^Kip1−/− bone marrow has a 5.7-fold increase in the frequency of stem cells, and surprisingly, an expanded pool of quiescent HSCs. However, Mad1^−/−p27^Kip1−/− stem cells exhibit an enhanced proliferative response under conditions of stress, such as cytokine stimulation in vitro and regeneration of the haematopoietic system after ablation in vivo. Together these data demonstrate that the MYC-antagonist MAD1 and cyclin-dependent kinase inhibitor p27^Kip1 cooperate to regulate the self-renewal and differentiation of HSCs in a context-dependent manner.