Primary patient-derived paediatric glioblastoma (pGBM) cells, cultured under conditions designed to maintain stem-like phenotypes, provide valuable platforms for pre-clinical research. Culturing tumour cells in suspension (3D) or adherent on laminin (2D) may influence their growth behaviour in vivo. Longitudinal, anatomical MRI showed that orthotopic HSJD-GBM-001 xenografts derived from 3D-cultured cells grew significantly faster than those from 2D-culture. Infiltrative tumour growth, maintenance of the blood brain barrier and high ADC, T1 and T2, associated with histopathologically confirmed tumour-oedema, were observed in both cohorts. The study highlights the influence of in vitro cell culture conditions on in vivo tumour growth characteristics.