Exploring effective, prompt and universally applicable approaches for inducing the differentiation of glioblastoma (GBM) into terminally differentiated cells, such as astrocytes or neurons that cease cell division, is pivotal for the success of GBM differentiation therapy. In this study, a neuronal-specific promoter-reporter system was employed to screen small molecules that promote neural differentiation. The cocktail YFSS, consisting of Y27632, Forskolin, SB431542 and SP600125, which selectively targets the ROCK, cAMP, TGF-β and JNK signalling pathways, respectively, was found to effectively trigger differentiation in human GBM cells. This process yielded neuron-like cells within 7 days, inhibited GBM cell proliferation and reduced malignancy traits, such as stemness, migratory and invasive capabilities. Transcriptome sequencing revealed the pathways altered by YFSS, shedding light on its dual role in halting cell proliferation and initiating neuronal differentiation. A notable increase in CEND1 expression, a key molecule in cell cycle and neuronal differentiation regulation, was observed during differentiation. However, CEND1 alone could not replicate YFSS's high conversion efficiency and its depletion reduced the differentiation and restored proliferation of the GBM cells. In vivo, prolonged and localised YFSS application significantly curtailed tumour growth and extended survival in patient-derived xenograft mice models. In summary, our findings reveal that the small-molecule cocktail YFSS is an effective means for inducing neuronal differentiation in GBM cells, representing a novel and promising pathway for the advancement of GBM treatment.