The enzyme NOS2 produces nitric oxide, a signaling molecule involved in processes like blood pressure regulation, immunity, and stem cell differentiation. NOS2 has three isoforms (NOS2-1, NOS2-2, and NOS2-3), but it’s unclear which one is expressed in differentiating human pluripotent stem cells (hPSCs), which are important tools for regenerative medicine and research. To learn more about NOS2 in these essential cells, researchers recently examined public RNA sequencing data for hPSCs. They found that NOS2 mRNA was transiently expressed during differentiation of hPSCs into various cell types, such as heart muscle, cartilage, and pre-placental cells, and the specific isoform expressed, NOS2-2, had a markedly different structure than the common immunity-related isoform NOS2-1. In vitro, four separate hPSC cell lines transiently expressed NOS2 mRNA and protein while they were differentiating into cortical neurons, and further analyses revealed that only the NOS2-2 isoform was expressed in these cells. Forced expression of this isoform in a cancer cell line confirmed that the resulting protein was functional. Although the mechanisms still need to be determined, the findings reveal a differentiation-specific NOS2 isoform in stem cells and provide insights that may help improve understanding of stem cells and related therapies.