In this study, we demonstrate that 5-FU activates CSCs via p53-induced WNT3 transcription, which is followed by activation of the WNT/β-catenin pathway in CRC cell lines and xenograft tumors as well as patient avatar models, such as patient-derived tumor organoids (PDTOs) and patient-derived cells (PDCs). Here, TP53 is linked to neoplasm.