Dendrimer is a composite nanomaterial with a diameter below 10 nm as a primary particle. This nanoparticle has unique properties such as sphere shape, wide variation in number of branches or surface radicals, and has been prospected of application to drug delivery, gene therapy and biological imaging. Our previous study showed that polyamidoamine (PAMAM) dendrimers were transported into cultured human neural progenitor cells (hNPCs) and were localized in lysosomal vesicles within the cells. In this study, we performed in vitro cytotoxicity tests for PAMAM dendrimers using hNPCs under 2D- and 3D-culture protocols evaluating neuronal differentiation. We added dendrimer nanoparticles (generation: 0 to 7; surface: PAMAM-NH2, PAMAM-OH, PAMAM-OS, PAMAM-SC, etc.) into media containing hNPCs with forms adhered to dishes or with forms of embryoid body (EB) in round-bottom wells, and maintained them for 3 days. After induction of differentiation for 4 additional days, we measured cell viability (MTT assay), apoptotic profiles (morphological analysis) and neurite expansion (morphological analysis). Our results indicate that amine-terminated PAMAM dendrimers affect neuronal cell functions in a dose-dependent manner, and that the effect might be related to cationically-charged surface of the particles.