The production of blood cells during steady-state and increased demand depends on the regulation of hematopoietic stem cell (HSC) self-renewal and differentiation. Similarly, the balance between self-renewal and differentiation of leukemia stem cells (LSCs) is crucial in the pathogenesis of leukemia. Here, we document that the TNF receptor superfamily member lymphotoxin-β receptor (LTβR) and its ligand LIGHT regulate quiescence and self-renewal of murine and human HSCs and LSCs. Cell-autonomous LIGHT/LTβR signaling on HSCs reduces cell cycling, promotes symmetric cell division and prevents primitive HSCs from exhaustion in serial re-transplantation experiments and genotoxic stress. LTβR deficiency reduces the numbers of LSCs and prolongs survival in a murine chronic myeloid leukemia (CML) model. Similarly, LIGHT/LTβR signaling in human G-CSF mobilized HSCs and human LSCs results in increased colony forming capacity in vitro. Thus, our results define LIGHT/LTβR signaling as an important pathway in the regulation of the self-renewal of HSCs and LSCs.