INTRODUCTION: Glioblastomas represent a considerable therapeutic challenge due to their remarkable heterogeneity and adaptability and potent ability to evade or resist current treatment strategies. Recent studies point to a small subset of cells – the cancer stem cell population – that may mediate many of the clinical and biological phenomenon associated with glioblastoma – for example, angiogenesis, immunosuppression, tumour self renewal etc. Inducing differentiation in the cancer stem cell population is an attractive approach which may not only eradicate much of the tumorigenic behaviour associated with these cells but render them more resistant to therapy. METHODS: Using an early passage biopsy-derived human paediatric cell line grown in neurobasal medium under normoxic conditions, we have treated the cells for 72 hours with either db-cAMP and theophylline (1mM), PACAP (1nM) or ATRA (10nM) before analysing the expression of GFAP, O4, Tu-20, CD133, Nestin, Mushashi 1, Olig 2 and Sox2 by flow cytometry and immunocytochemistry. Viability, proliferation, live cell imaging and cell cycle analyses were also carried out. RESULTS: Of the three agents, db-cAMP and theophylline had the most striking effect, with cells differentiating along an astrocytic lineage within 48 hours as shown by the increased expression of GFAP and down-regulation of the stem cell markers. Treatment with ATRA and PACAP showed similar but less pronounced effects (where p = 0.05). CONCLUSIONS: Differentiation agents, in particular db-cAMP and theophylline, appear to reduce the stem cell like behaviour of glioblastoma cells treated in vitro. Future studies will include elucidating signal transduction pathways as well as determining post-differentiation therapeutic sensitivity to nuclear and mitochondrial agents.