Abstract In mouse blastocysts six facilitative glucose transporter isoforms (GLUT)1–4, 8 and 9 are expressed. We have used the mouse embryonic stem (ES) cell line D3 and spontaneously differentiating embryoid bodies (EB) to investigate GLUT expression and the influence of glucose during differentiation of early embryonic cells. Both ES cells and EBs (2d–20d) expressed GLUT1, 3, and 8, whereas the isoforms 2 and 4 were detectable exclusively in EBs. Differentiation-associated expression of GLUT was analyzed by double staining with stage-specific embryonic antigen (SSEA-1), cytokeratins (CK18, 19), nestin, and desmin. Similar to trophoblast cells in mouse blastocysts the outer cell layer of endoderm-like cells showed a high GLUT3 expression in early EBs. In 20-day-old EBs no GLUT3 protein and only minor GLUT3 mRNA amounts could be detected. A minimal glucose concentration of 5 mM applied during 2 and 8 days of EB culture resulted in up-regulated GLUT4, Oct-4 and SSEA-1 levels and a delay in EB differentiation. We conclude that GLUT expression depends on cellular differentiation and that the expression is modulated by glucose concentration. The developmental and glucose-dependent regulation of GLUT strongly suggests a functional role of glucose and glucose transporters in ES cell differentiation and embryonic development.