Pluripotent stem cells (PSCs) are characterized by the ability to self-renew as well as undergo multidirectional differentiation. Culture conditions have a pivotal influence on differentiation pattern. In the current study, we compared the fate of mouse PSCs using two culture media: (1) chemically defined, free of animal reagents, and (2) standard one relying on the serum supplementation. Moreover, we assessed the influence of selected regulators (WNTs, SHH) on PSC differentiation. We showed that the differentiation pattern of PSCs cultured in both systems differed significantly: cells cultured in chemically defined medium preferentially underwent ectodermal conversion while their endo- and mesodermal differentiation was limited, contrary to cells cultured in serum-supplemented medium. More efficient ectodermal differentiation of PSCs cultured in chemically defined medium correlated with higher activity of SHH pathway while endodermal and mesodermal conversion of cells cultured in serum-supplemented medium with higher activity of WNT/JNK pathway. However, inhibition of either canonical or noncanonical WNT pathway resulted in the limitation of endo- and mesodermal conversion of PSCs. In addition, blocking WNT secretion led to the inhibition of PSC mesodermal differentiation, confirming the pivotal role of WNT signaling in this process. In contrast, SHH turned out to be an inducer of PSC ectodermal, not mesodermal differentiation.