© 2018 The Authors Background: Differentiation of human embryonic stem cells (hESCs) into distinct neural lineages has been widely studied. However, preparation of mixed yet neurochemically mature populations, for the study of neurological diseases involving mixed cell types has received less attention. New method: We combined two commonly used differentiation methods to provide robust and reproducible cultures in which a mixture of primarily GABAergic and Glutamatergic neurons was obtained. Detailed characterisation by immunocytochemistry (ICC) and quantitative real-time PCR (qPCR) assessed the neurochemical phenotype, and the maturation state of these neurons. Results: We found that once neurospheres (NSs) had attached to the culture plates, proliferation of neural stem cell was suppressed. Neuronal differentiation and synaptic development then occurred after 21 days in vitro (DIV). By 49DIV, there were large numbers of neurochemically and structurally mature neurons. The qPCR studies indicated that expression of GABAergic genes increased the most (93.3-fold increase), followed by glutamatergic (51-fold increase), along with smaller changes in expression of cholinergic (3-fold increase) and dopaminergic genes (6-fold increase), as well as a small change in glial cell marker expression (5-fold increase). Comparison with existing method (s): Existing methods isolate hESC-derived neural progenitors for onward differentiation to mature neurons using either migration or dissociative paradigms. These give poor survival or yield. By combining these approaches, we obtain high yields of morphologically and neurochemically mature neurons. These can be maintained in culture for extended periods. Conclusion: Our method provides a novel, effective and robust neural culture system with structurally and neurochemically mature cell populations and neural networks, suitable for studying a range of neurological diseases from a human perspective.