Neurons derived from human embryonic stem cells (hESCs) represent an inexhaustible source of cells for transplantation in neurodegenerative disease, for modelling cell physiology and for drug discovery programs. In Parkinson’s disease (PD), midbrain dopaminergic neurons of the A9 group are progressively lost which leads to the classic motor symptoms of PD. Our lab has recently created a human stem cell reporter line expressing eGFP under the control of the specific midbrain dopaminergic transcription factor, PITX3, allowing us to identify these neurons as they differentiate. In contrast to the large amount of research directed towards optimising differentiation conditions, the functional properties of neurons derived from hESCs remain poorly understood. In this study we asked whether our PITX3-eGFP+ cells responded to common neurotransmitters present in the midbrain, and whether these responses changed during differentiation. Cells were differentiated in monolayer according to an efficient protocol published recently1, and imaged on days 20, 40, 60, and 80. At day 20 of differentiation, PITX3-eGFP+ neurons responded to γ-amino butyric acid (GABA), noradrenaline (NA), L-glutamate (Glut), acetylcholine (ACh; all 30µM), adenosine triphosphate (ATP; 300µM), KCl (30mM), and Ca2+ (20mM) with elevations of [Ca2+]i. The magnitude of Glut, ATP, KCl, and Ca2+ induced elevations increased significantly by day 80, at the same time as GABA induced elevations decreased, indicating functional development during differentiation (one-way ANOVA with post hoc Dunnett’s test, P<0.05, n=4). From day 20 to 80 basal [Ca2+]i increased from 44.57 ± 12.99 nM to 168.67 ± 25.2 nM, while basal [Cl-]i decreased from 5.70 ± 0.35 mM to 2.18 ± 0.39 mM. Our results show that PITX3-eGFP+ neurons derived from human ESCs recapitulate some key functional aspects of midbrain dopaminergic neurons, and that functional development continues for some time following the appearance of post-mitotic, “mature” neuron transcription factors, such as PITX3.