Mouse embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells can be used as models of neuronal differentiation for the investigation of mammalian neurogenesis, pharmacological testing, and development of cell-based therapies. Recently, mouse iPS cell lines have been generated by Sleeping Beauty (SB) transposon-mediated transgenesis (SB-iPS). In this study, we determined for the first time the differentiation potential of mouse SB-iPS cells to form neuronal progenitor cells (NPCs) and neurons. Undifferentiated SB-iPS and ES cells were aggregated into embryoid bodies (EBs) and cultured in neuronal differentiation medium supplemented with 5 μM all-trans retinoic acid. Thereafter, EBs were dissociated and plated to observe further neuronal differentiation. Samples were fixed on days 10 and 14 for immunocytochemistry staining using the NPC markers Pax6 and Nestin and the neuron marker βIII-tubulin/Tuj1. Nestin-labeled cells were analyzed further by flow cytometry. Our results demonstrated that SB-iPS cells can generate NPCs and differentiate further into neurons in culture, although SB-iPS cells produced less nestin-positive cells than ESCs (6.12 ± 1.61 vs. 74.36 ± 1.65, respectively). In conclusion, the efficiency of generating SB-iPS cells-derived NPCs needs to be improved. However, given the considerable potential of SB-iPS cells for drug testing and as therapeutic models in neurological disorders, continuing investigation of their neuronal differentiation ability is required.