Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are very attractive cell sources for the treatment of diabetes mellitus, because numerous cells can be obtained using their infinite proliferation potential to overcome the paucity of donor islets. Advances in differentiation protocols make it possible to generate glucose responsive hPSC-beta cells, which can ameliorate hyperglycemia in diabetic mice. These protocols have mainly been based on an adherent culture system. However, in clinical applications, suspension culture methods are more suitable for large-scale culture. There are reports that suspension culture and spheroid formation promote differentiation in various cell types, including hPSCs, but, to our knowledge, there are no reports comparing gene expression patterns between suspension and adherent cultured human iPSCs (hiPSCs) during definitive endoderm (DE) differentiation. In this study, we chose several stage marker genes, not only for DE but also for posterior epiblast and primitive streak, and we examined their time course expression in suspension and adherent cultures by quantitative PT-PCR (qPCR), western blot, flow cytometry and immunocytochemistry. Our results demonstrate that expressions of these marker genes are faster and more strongly induced in suspension culture than in adherent culture during the DE differentiation process, indicating that suspension culture favors DE differentiation.