Mouse embryonic stem cell (mESC)-derived neurons are a renewable cell source for investigation of neuronal circuits. Engineering circuit-tracing components into stem cells facilitates studies on mechanisms of synaptic coupling and circuitogenesis. This unit details methods for the generation of mESC-derived neurons harboring trans-synaptic viral tracing elements, which are used for investigation of synaptic connections within circuits in vitro, ex vivo, and in vivo. The first protocol describes procedures for feeder-free passaging of mESCs, modified to carry reporter and rabies virus tracing elements. The second protocol describes in vitro generation of neurons from these ESCs. The last protocols describe the use of ESC-derived neurons as "source cells" for rabies virus circuit-tracing to identify inputs onto synaptically connected neurons. Given the broad applicability, these protocols can be applied to investigate the ability of in vitro-derived neurons to establish/maintain synaptic connections in disease models, and/or with human-induced pluripotent stem cells.