Cytokine mediated inflammation likely plays an important role in secondary pathology after traumatic brain injury (TBI). The aim of this study was to elucidate secondary cytokine responses in an in vitro enriched (>80%) human stem cell-derived neuronal model. We exposed neuronal cultures to pre-determined and clinically relevant pathophysiological levels of tumor necrosis factor-α (TNF), interleukin-6 (IL-6) and interleukin-1β (IL-1β), shown to be present in the inflammatory aftermath of TBI. Data from this reductionist human model were then compared with our in vivo data. Human embryonic stem cell (hESC)-derived neurons were exposed to recombinant TNF (1-10,000 pg/mL), IL-1β (1-10,000 pg/mL), and IL-6 (0.1-1000 ng/mL). After 1, 24, and 72 h, culture supernatant was sampled and analyzed using a human cytokine/chemokine 42-plex Milliplex kit on the Luminex platform. The culture secretome revealed both a dose- and/or time-dependent release of cytokines. The IL-6 and TNF exposure each resulted in significantly increased levels of >10 cytokines over time, while IL-1β increased the level of C-X-C motif chemokine 10 (CXCL10/IP10) alone. Importantly, these patterns are consistent with our in vivo (human) TBI data, thus validating our human stem cell-derived neuronal platform as a clinically useful reductionist model. Our data cumulatively suggest that IL-6 and TNF have direct actions, while the action of IL-1β on human neurons likely occurs indirectly through inflammatory cells. The hESC-derived neurons provide a valuable platform to model cytokine mediated inflammation and can provide important insights into the mechanisms of neuroinflammation after TBI.