The high grade gliomas (HGG) are a collection of lethal brain tumours. Currently available therapies are unsuccessful and patient survival rates have not significantly improved over the last ~ 50 years. Preclinical models currently in use poorly predict subsequent activity in phase I trials and over-estimate anti-tumour activity in HGG. Brain tissue provides a unique physical and biochemical tumour microenvironment, when compared with the microenvironment of other solid tumours. The incorporation of tissue dimensionality and healthy brain tissue mimetics into preclinical assessments may thus improve preclinical assessments, providing a more accurate reflection of in vivo response. In the present study we have conducted evaluations of Ephrin receptor A2 (EphA2)-directed CAR T cells in paediatric brain cancer, comparing response in target cells grown in 2D cultures, as 3D spheroids and as 3D spheroids co-cultured with stem-cell derived cortical brain organoids. EphA2-CAR T cell induced cytotoxicity under 2D conditions was assessed by xCelligence electrical impedance assay and cytokine release confirmed by ELISA. CAR T efficacy under 3D conditions was assessed in tumour spheroids and tumour spheroid/brain organoid co-cultures using luciferase reporters and the IVIS Lumina in vivo imaging system. As predicted, CAR T cell efficacy reduced with increasing model complexity. There was no direct correlation between EphA2 cell surface expression and CAR T mediated cell death, with some cells displaying high EphA2 expression being less sensitive to CAR T treatment under 3D conditions. Under 3D assay conditions, we detected differences in the efficacy of CAR T cells prepared from different donors, with the greatest differences seen in the spheroid/brain organoid co-cultures. Collectively the data reveal that the CAR T cells exhibit less efficacy when assessed in 3D models. We contend that this likely better reflects the in vivo response. The use of such biomimetic models may not only provide a more realistic reflection of in vivo responses but also gives us an opportunity to develop robust assay platforms to increase successful transition of treatments to patients.