The flt3 ligand (FL) is a growth factor for primitive hematopoietic cells. Serum levels of FL are inversely related to the number and proliferative capacity of early hematopoietic progenitors. We sought to elucidate the molecular mechanism underlying this regulation. Expression of FL was examined in peripheral blood (PB) and bone marrow (BM) cells under normal steady-state hematopoiesis and during transient BM failure induced by chemoradiotherapy in 16 patients with hematological malignancies. Using anti-FL antibodies in Western analysis, flow cytometry, and confocal microscopy, we detected high levels of preformed FL inside but not on the surface of T lymphocytes in steady-state hematopoiesis. Intracellular FL colocalized with giantin and ERGIC-53, indicating that it is stored within and close to the Golgi apparatus. After chemotherapy-induced hematopoietic failure, FL rapidly translocated to the surface of T lymphocytes and the levels of FL released to serum increased approximately 100-fold. Expression of FL mRNA was enhanced only about sevenfold; a similar, twofold to sixfold increase in mRNA was observed in the thymus and BM of mice with irradiation-induced aplasia. Upregulation of FL mRNA was delayed when compared with the appearance of cell surface-associated and soluble protein isoforms. The described changes in FL expression in response to chemotherapy-induced aplasia were observed in all patients, irrespective of the diagnosis and treatment regimen. Our data demonstrate that mobilization of preformed FL from intracellular stores rather than de novo synthesis is responsible for increased FL levels in BM failure.