Pancreatic ductal adenocarcinoma (PDAC) is a major histological type of pancreatic cancer and remains one of the most lethal cancers with a high mortality rate owing to its aggressive growth, high metastatic rate, and recurrence. Recent studies on cancer stem cells (CSCs) have suggested that the aggressive growth, high metastatic rate, and recurrence might be caused by the ability of CSCs to self-renew, differentiate, and drive tumorigenesis. Thus, CSCs are expected to be a therapeutic target for PDAC. Sphere forming assay of cancer cells, including PDAC cells, is commonly performed using epidermal growth factor and fibroblast growth factor-2 containing serum-free medium to identify and isolate the enriched CSCs. Recently, we observed that PDAC spheres cultured in fetal bovine serum containing medium are morphologically similar to spheres cultured in the growth factor containing medium. In this study, we cultured two PDAC cell lines, PANC-1 and PK-1, in growth factor containing serum-free medium or serum containing medium, and compared the morphology of the spheres formed in detail by electron microscopy and examined the expression of major CSC marker genes. Both the PDAC cells formed larger spheres in the serum containing medium than in the growth factor containing medium. PK-1 cells formed more morphologically differentiated spheres, with peripheral flat lining cells, in the serum containing medium. Expression levels of most of the CSC markers were higher in the spheres of the two PDAC cells in both the culture mediums than in the cells cultured under adherent conditions. The expression levels of CSC markers in PDAC spheres cultured in the growth factor containing medium were not necessarily higher than that in the spheres cultured in the serum containing medium. These findings suggest that sphere forming assay using serum containing medium, by which large PDAC spheres with enriched CSCs are formed, may be useful for deciphering the characteristics of CSCs and for developing anti-CSC therapies for PDAC.