Signaling-pathway analyses and the investigation of gene responses to different stimuli are usually performed in 2D monocultures. However, within the glomerulus, cells grow in 3D and are involved in direct and paracrine interactions with different glomerular cell types. Thus, the results from 2D monoculture experiments must be taken with caution. We cultured glomerular endothelial cells, podocytes and mesangial cells in 2D/3D monocultures and 2D/3D co-cultures and analyzed cell survival, self-assembly, gene expression, cell–cell interaction, and gene pathways using live/dead assay, time-lapse analysis, bulk-RNA sequencing, qPCR, and immunofluorescence staining. Without any need for scaffolds, 3D glomerular co-cultures self-organized into spheroids. Podocyte- and glomerular endothelial cell-specific markers and the extracellular matrix were increased in 3D co-cultures compared to 2D co-cultures. Housekeeping genes must be chosen wisely, as many genes used for the normalization of gene expression were themselves affected in 3D culture conditions. The transport of podocyte-derived VEGFA to glomerular endothelial cells confirmed intercellular crosstalk in the 3D co-culture models. The enhanced expression of genes important for glomerular function in 3D, compared to 2D, questions the reliability of currently used 2D monocultures. Hence, glomerular 3D co-cultures might be more suitable in the study of intercellular communication, disease modelling and drug screening ex vivo.