Various types of embryoid body (EB) that were formed from mouse embryonic stem (ES) cells under various culture conditions were characterized in terms of gene expression pattern to estimate the differentiation status of the bodies. The gene expression of typical markers (i.e., GATA-4, GATA-6, transthyretin [TTR], alpha-fetoprotein [AFP], Nkx2.5, and alpha-myosin heavy chain [alpha-MHC]) was quantitatively analyzed in various types of EB, and the gene expression pattern of those marker genes was graphically shown for each EB. The gene expression pattern accurately represented the differentiation status of the EBs. The gene expression pattern indicated that the Nkx2.5 and alpha-MHC genes were highly expressed in the EBs formed from 1000 ES cells in a low-adherence 96-well plate. By transferring the EBs into an attachment culture, cardiomyocytes were more efficiently generated in the outgrowth of the EBs. When we increased the seeding cell number from 1000 to 4000 ES cells, the gene expression pattern changed, that is, the expression levels of the TTR and AFP genes increased, whereas those of the Nkx2.5 and alpha-MHC genes decreased, and the trend of differentiation changed from cardiomyogenesis to visceral yolk-sac-like structure formation.