Maintenance of glucose uptake is a key component in the response of hematopoietic cells to survival factors. To investigate the mechanism of this response we employed the interleukin-3 (IL-3)-dependent murine mast cell line IC2.9. In these cells, hexose uptake decreased markedly upon withdrawal of IL-3, whereas its readdition led to rapid (t1/2 ∼ 10 min) stimulation of transport, associated with an ∼4-fold increase in Vmax but no change in Km. Immunocytochemistry and photoaffinity labeling revealed that IL-3 caused translocation of intracellular GLUT1 transporters to the cell surface, whereas a second transporter isoform, GLUT3, remained predominantly intracellular. The inhibitory effects of latrunculin B and jasplakinolide, and of nocodazole and colchicine, respectively, revealed a requirement for both the actin and microtubule cytoskeletons in GLUT1 translocation and transport stimulation. Both IL-3 stimulation of transport and GLUT1 translocation were also prevented by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002. The time courses for activation of phosphatidylinositol 3-kinase and its downstream target, protein kinase B, by IL-3 were consistent with a role in IL-3-induced transporter translocation and enhanced glucose uptake. We conclude that one component of the survival mechanisms elicited by IL-3 involves the subcellular redistribution of glucose transporters, thus ensuring the supply of a key metabolic substrate.