Abstract BACKGROUND Glioblastoma (GBM) is the most prevalent and lethal primary brain tumor. Its recurrence and resistance to current therapies, i.e. temozolomide (TMZ), is thought to result from a subpopulation of cells exhibiting stem cell properties, called glioblastoma stem-like cells (GSCs). Bone Morphogenetic Protein 4 (BMP4) induces GSCs differentiation, leading to a less resistant phenotype. In this study we show that co-treatment of BMP4 with TMZ has therapeutic benefit in a subset of GBM cell lines. Furthermore, we looked for patients’ molecular signatures that could predict sensitivity to this combination treatment. MATERIAL AND METHODS A panel of 17 primary GBM cultures (passages 5–10) were treated with increasing concentrations of TMZ, BMP4 and TMZ + BMP4 for 5 and 7 days and cell viability has been measured. The Combination Index (CI) of the two drugs was calculated to assess the response of each line to TMZ and BMP4 treatment. DNA was used to determine the MGMT promotor methylation status and for targeted exome sequencing. Expression levels of BMP signaling components and differentiation associated genes were determined by qPCR. RESULTS 12 cultures of primary GBMs and 5 cultures of recurrent GBMs were included. Overall, 71% of the tested cell lines was resistant to TMZ, while 41% was resistant to BMP4. Strikingly, 53% of primary cultures show synergy between TMZ and BMP4 (CI < 1 at a Fraction Affected of 50% (Fa50)). There was no significant difference in synergy between five or seven days of treatment. However, combination treatment of BMP4 and TMZ was more effective than sequential treatment. CONCLUSION Co-treatment of BMP4 and TMZ could be of therapeutic benefit in GBM patients, irrespective of their sensitivity to TMZ. Further research regarding the mechanism behind this synergy is necessary as to identify predictive markers for treatment response.