Embryoid body (EB) formation is an important step in the differentiation of pluripotent stem cells. Although glucose concentration is physiologically maintained at 5.5 mM (low glucose; LG) in vivo, a medium containing 25 mM glucose (high glucose; HG) has been widely used for forming EBs in vitro. In this study, we investigated the effect of glucose concentration during EB formation from mouse embryonic stem (ES) cells on EB growth and cell differentiation. EBs were formed under various glucose concentrations: 40, 25, 5.5, and 0 mM. Cells aggregated to form EBs regardless of glucose concentration, but 0 mM glucose was not appropriate for supporting EB growth as compared with 25 mM glucose. The EBs that formed in the presence of 5.5 mM glucose (LG-EBs) were similar both in terms of appearance and decreased expression levels of undifferentiated-ES-cell-marker genes to the EBs that formed in the presence of 25 mM glucose (HG-EBs). However, there was a difference in the propensity for cell differentiation between LG-EBs and HG-EBs. In directed differentiation cultures of EBs into cardiomyocytes and neuronal cells, the HG-EBs more efficiently generated beating cardiac muscle, and the LG-EBs more specifically generated beta III-tubulin-positive cells. These findings demonstrate that the high-glucose (25 mM) condition was not necessary for EB formation in mouse ES cells, whereas the glucose concentration during EB formation affects the propensity for cell differentiation in the attachment cultures of formed EBs. The physiological low-glucose (5.5 mM) condition was suitable for forming EBs directed toward neuronal cell differentiation in mouse ES cells. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.