Pediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are rare, but rapidly fatal malignancies of the central nervous system (CNS), and the leading cause of cancer-related death in children. Besides the scarcity of available biological material for research, the study of these diseases has been hampered by methodological problems. One of the major obstacles is the difficulty with which these cells can be genetically modified by conventional laboratory methods, such as lentiviral transduction. As a result, only very few successful stable modifications have been reported. As pHGG and DIPG cells are most often cultured as neurospheres, and therefore retain stem cell-like characteristics, we hypothesized that this culture method is also responsible for their resistance to transduction. We therefore developed a protocol in which pHGG and DIPG cells are temporarily forced to form an adherent monolayer by exposure to serum, to create a window-of-opportunity for lentiviral transduction. We here demonstrate that this protocol reliably and reproducibly introduces stable genetic modifications in primary DIPG and pHGG cells. • Short-term serum exposure enables lentiviral transduction of primary pediatric glioma neurospheres.