Cyanobacterial blooms are increasing in frequency and intensity globally, and impacting recreational waters as well as waters used for drinking water provisioning. They are sources of bioactive metabolites including retinoids and the neurotoxin anatoxin-a. Here, we investigated the effects of anatoxin-a on a differentiating in vitro human neural stem cell model previously characterised with retinoic acids. Effects on protein and gene expression upon exposure for 9 or 18 days to anatoxin-a alone or in co-exposure with all-trans retinoic acid were evaluated using a panel of neural and glial differentiation biomarkers. Anatoxin-a did not cause distinct developmental neuro-toxicity alone, or in co-exposure with retinoic acid. However, in line with its excitotoxicity, in co-exposure with 200 nM all-trans retinoic acid it reduced the differentiation of acetylcholinergic neuron subtypes in the culture at 1000 nM (highest tested concentration). While this could have substantial functional implications for the developing nervous system, there is no indication for developmental neurotoxicity beyond its (excito-)toxicity to acetylcholinergic neurons, which only occurred in co-exposure to all-trans retinoic acid.