Transient protein expression can easily be studied in i3Neurons using lipid-based transfection. This protocol is identical to that in iPSCs (see Basic Protocol 2). i3Neurons are modestly transfectable, with 5 % to 10 % of cells showing fluorescent protein expression after 24 hr. We have found that refreshing neuronal medium 1 to 2 hr after transfection both allows successful DNA entry into cells and largely prevents cytotoxicity resulting from the transfection reagent. Unlike iPSCs, i3Neurons show increased protein expression\/accumulation over time, with greater fluorescence 48 to 72 hr after transfection than at 24 hr. Transient transfections also show more durable expression in i3Neurons than iPSCs, likely because episomes are not diluted by cell division. i3Neurons can be transfected in suspension (i.e., re-plating after day 3 of differentiation) or as an adherent culture, although better results are observed in adherent cultures. They are also amenable to serial transfections (i.e., re-transfecting with the same construct 24 hr apart) if higher-percentage transfections are desired.