The ability to generate regionally defined neuronal populations from human pluripotent stem cells (hPSCs) in vitro provides an increasingly utilised experimental resource for the investigation of human neuronal physiology and neurological disorders. Despite this, the physiological properties of such neurones have remained understudied. In this regard, we have developed a protocol that efficiently generates populations of excitatory cortical neurones derived from hPSCs (hECNs) and examined the functional properties of these neurones with respect to previously determined regionally-specific features and, importantly, well-defined foetal and adult properties in their native mammalian counterparts. To generate hECNs, in vitro embryonic or induced hPSCs are neuralised using an approach based upon the default model of neurogenesis that minimizes extrinsic and intrinsic signals that lead to alternative cell fates (Bilican et al. 2014). Neural stem cells (NSCs) generated with this protocol express anterior brain markers FOXG1 and OTX2. When NSCs are dissociated and plated as monolayer they terminally differentiate to an enriched population of VGLUT1+-hCENs that express cortical layer markers CTIP2, SAT2B or Reelin. Glia and GABAergic interneurones account for <10% of the cell population. Electrophysiological assessment of the intrinsic properties of hECNs indicates that they become progressively more excitable with time; after 5 weeks in culture >95% of cells fire action potentials. During this period of maturation we also observe that hECNs show a reduction in their input resistance and an increase in whole-cell capacitance in association with changes in excitability.. To ascertain whether hECNs expressed ligand-gated ion channels (LGICs) with immature- or mature-like properties, we carried out both biophysical and pharmacological characterisation of major neurotransmitter LGICs. Our analysis indicates that hECNS express GABAARs with a likely subunit combination of α2/3β3γ2, (James et al. 2014) while the majority of NMDARs are diheteromeric assemblies of GluN1 and GluN2B subunits. In the rodent cortex these combinations are predominately associated with early postnatal development. Interestingly our data indicate that for AMPARs, hECNs express subunit combinations that are more typical of a mature adult cortex (Livesey et al. 2014). In the mammalian CNS, the developmentally-regulated GluA2 subunit is subjected to post-transcriptional editing where a M2 pore-lining glutamine (Q) codon is edited to arginine (R), which imparts a reduced single-channel conductance (γ), insensitivity to polyamine block and reduced Ca2+-permeability to the AMPAR complex. Measurements of AMPAR γ in hECNs at week 2 (~11 pS) and week 5 (~4 pS) are consistent with a developmental shift from GluA2(R)-lacking to GluA2(R)-containing AMPARs. Consistent with changes in unitary conductance, AMPAR-mediated currents recorded from Week 5 hECNs are insensitive to polyamine block. Interestingly, an equivalent upregulation of GluA2(R)-containing AMPARs over an equivalent time period is observed when human cortical neurones are differentiated in vitro from native human foetal cortical NPCs suggesting that the human AMPAR development time-course maybe distinct from that of seen in rodents. Using a similar experimental approach, data will be presented showing that motor neurones derived from hPSCs exhibit a rapid up regulation of the GluA2-edited AMPAR in vitro. The editing of AMPARs is a major focus for motor neurone disease (MND) research where human motor neurones show abnormalities in AMPAR editing to yield an increase in Ca2+-permeable GluA2(Q)-containing AMPARs that are thought to give rise to glutamate-mediated excitotoxicity in affected adult neurones. Data investigating functional AMPAR regulation in cortical and motor neurones derived from MND patients will be presented. In vitro hPSC-derived neurones offer tremendous opportunities to gain mechanistic insight into human neurological disorders however it has become apparent from our own and other’s work that the neurophysiological properties of such neurones need to be characterised carefully in order to exploit them to their full potential in studies disease-modelling.