Colorectal cancer (CRC) is one of the most common solid organ cancers prevalent worldwide causing, in spite of advancing therapeutic methodology, high rate of patient mortality, especially due to metastasis development. The cancer stem cell (CSC) theory of tumor growth indicates that CSCs within the tumor mass have great capacity to initiate and sustain tumor growth. Following the suggestion that Fas signaling can be engaged in apoptosis, tumor maintenance, senescence or DICE (death induced by CD95 or CD95L elimination), the attempts to broaden the knowledge concerning the relationships between CSCs features and FasR/FasL appeared to be necessary. The most important advantage of our study was the simultaneously analysis of CSCs from commonly used CRC lines (HCT116 and HT29) and tumor fragments collected from CRC patients. Moreover, the sphere-promoting expansion of CRC lines brought a specific three-dimensional specific environment for CSC exploration. We further investigated the function of Fas signaling in CRC lines depending on the culture mode as we incubated HCT116 and HT29 cells with anti-FasR agonistic antibodies. It appeared to act in a line-dependent and culture mode-dependent manner and influenced some particular features of CSCs such as spherogenicity, proliferation and phenotype. Additionally, the analysis of mRNA level showed that disease progression is associated with significantly increased expression of FasR and/or FasL. In conclusion, our observation seems to confirm that spherical model of cancer lines is more reliable for some sophisticated analysis because of their greater resemblance to the CSCs from human CRC samples in comparison to commonly used adherent cells, at least according to aspects of their biology analyzed in this study. That can be extended to the resemblance of in vitro sphere forming conditions to the in vivo environment. However, the greatest difference concerns the level of apoptosis, thus, this issue require further experiments.