The role of acidic glycosphingolipids in cell growth and differentiation was investigated using the multipotent leukemia cell line K562. When GM₃ was added to cell culture media, the growth of K562 cells was remarkably inhibited and the cells were shown to have megakaryocytoid morphology. Ultrastructural study demonstrated that K562 cells treated with GM₃ had platelet peroxidase-positive structures, which were considered to be the specific marker of megakaryocyte. Furthermore, AP-3 directed against an epitope present on membrane glycoprotein IIIa reacted with the GM₃-treated cells. Free N-acetylneuraminic acid, GM₁, GM₂, GD_1a, and a mixture of bovine brain gangliosides containing GD_1a and GT_1b did not affect growth of K562 cells or show morphological changes. According to chemical analyses, GM₃ content increased in megakaryocytoid differentiation induced by tetradecanoylphorbol-13-acetate, whereas GM₃ decreased in erythroid differentiation induced by hemin. Enzymatic analysis showed that the GM₃ increase during megakaryocytoid differentiation was a result of the sialyltransferase activation. These results indicated that exogenous GM₃ induced differentiation of K562 cells into a “GM₃-rich” lineage, i.e., mainly megakaryocytoid lineage, and that GM₃ accumulation in the GM₃-rich lineage was the result of the activation of GM₃ synthase. These findings strongly suggested that GM₃ ganglioside, a minor membrane component, has a crucial role in not only the differentiation induction but also the determination of the differentiation direction in pluripotent K562 cells.