As the number of nanoparticle-based products increase in the marketplace, there will be increased potential for human exposures to these engineered materials throughout the product life cycle. We currently lack sufficient data to understand or predict the inherent nanomaterial characteristics that drive nanomaterial-biological interactions and responses. In this study, we utilized the embryonic zebrafish (Danio rerio) model to investigate the importance of nanoparticle (NP) surface functionalization, in particular as it pertains to nanoparticle stability, on in vivo biological responses. This is a comparative study where two lead sulfide nanoparticles (PbS-NPs) with nearly identical core sizes, but functionalized with either sodium 3-mercaptopropanesulfonate (MT) or sodium 2,3-dimercaptopropanesulfonate (DT) ligand, were used. Developmental exposures and assessments revealed differential biological responses to these engineered nanoparticles. Exposures beginning at 6 h post fertilization (hpf) to MT-functionalized nanoparticles (PbS-MT) led to 100% mortality by 120 hpf while exposure to DT-functionalized nanoparticles (PbS-DT) produced less than a 5% incident in mortality at the same concentration. Exposure to the MT and DT ligands themselves did not produce adverse developmental effects when not coupled to the NP core. Following exposure, we confirmed that the embryos took up both PbS-MT and PbS-DT material using inductively coupled plasma-mass spectrometry (ICP-MS). The stability of the nanoparticles in the aqueous solution was also characterized. The nanoparticles decompose and precipitate upon exposure to air. Soluble lead ions were observed following nanoparticle precipitation and in greater concentration for the PbS-MT sample compared to the PbS-DT sample. These studies demonstrate that in vivo assessments can be effectively used to characterize the role of NP surface functionalization in predicting biological responses.