The progression of colorectal cancer (CRC) is supposedly driven by cancer stem cells (CSC) which are able to self-renew and simultaneously fuel bulk tumour mass with highly proliferative and differentiated tumour cells. However, the CSC-phenotype in CRC is unstable and dependent on environmental cues. Fibroblast growth factor 2 (FGF2) is essential and necessary for the maintenance of self-renewal in adult and embryonic stem cells. Investigating its role in self-renewal in advanced CRC patient-derived organoids, we unveiled that FGF-receptor (FGFR) inhibition prevents organoid formation in very early expanding cells but induces cyst formation when applied to pre-established organoids. Comprehensive transcriptome analyses revealed that the induction of the transcription factor activator-protein-1 (AP-1) together with MAPK activation was most prominent after FGFR-inhibition. These effects resemble mechanisms of an acquired resistance against other described tyrosine kinase inhibitors such as EGF-receptor targeted therapies. Furthermore, we detected elevated expression levels of several self-renewal and stemness-associated genes in organoid cultures with active FGF2 signalling. The combined data assume that CSCs are a heterogeneous population while self-renewal is a common feature regulated by distinct but converging pathways. Finally, we highlight FGF2 signalling as one of numerous components of the complex regulation of stemness in cancer.