Human embryonic stem cells (hESCs) have been thought to be a promising cell source for cell transplantation therapy because they are self-renewing cells and can differentiate to all cell types derived from 3 embryonic germ layers. hESCs free from the risk of unknown transmitted pathogens are preferable for clinical use. In this study, we examined the combination of serum-free conditioned medium of human mesenchymal stem cells (hMSCs) and surfaces coated with recombinant attachment factors, ProNectin F Plus, for the embryonic stem cell (ESC) culture system using cynomolgus monkey ES (cESCs). After 22 passages, cESCs still expressed the pluripotent stem cell markers stage-specific embryonic antigen-4 and Oct3/4 and maintained the ability to form teratoma-containing tissues of all 3 embryonic germ layers in severe combined immunodeficiency mice. Various characteristics of cESCs have been reported to be similar to those of hESCs. Thus, the results obtained by using cESCs may be applied to studies of hESCs. We hypothesized that a combination of conditioned medium of hMSCs and a ProNectin F Plus-coated dish would afford a culture system for undifferentiated hESCs.