Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) coined by inflammation and neurodegeneration. The actual cause of the neurodegenerative component of the disease is however unclear. We investigated here the direct and differential effects of inflammatory mediators on human neurons. We used embryonic stem cell-derived (H9) human neuronal stem cells (hNSC) to generate neuronal cultures. Neurons were subsequently treated with tumour necrosis factor alpha (TNFα), interferon gamma (IFNγ), granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin 17A (IL-17A) and interleukin 10 (IL-10) separately or in combination. Immunofluorescence staining and quantitative polymerase chain reaction (qPCR) were used to assess cytokine receptor expression, cell integrity and transcriptomic changes upon treatment. H9-hNSC-derived neurons expressed cytokine receptors for IFNγ, TNFα, IL-10 and IL-17A. Neuronal exposure to these cytokines resulted in differential effects on neurite integrity parameters with a clear decrease for TNFα- and GM-CSF-treated neurons. The combinatorial treatment with IL-17A/IFNγ or IL-17A/TNFα induced a more pronounced effect on neurite integrity. Furthermore, combinatorial treatments with two cytokines induced several key signalling pathways, i.e. NFκB-, hedgehog and oxidative stress signalling, stronger than any of the cytokines alone. This work supports the idea of immune-neuronal crosstalk and the need to focus on the potential role of inflammatory cytokines on neuronal cytoarchitecture and function.