Culture of mouse zygotes at low density to the hatching blastocyst stage in medium containing selected small molecules improved development from the 5-cell stage through to the hatching blastocyst stage. Here, we show that these factors reduce mitochondrial membrane potential, reduce reactive oxygen species (ROS) levels, and increase glutathione (GSH) levels. Mitochondrial membrane potential and reactive oxygen species (ROS) levels in 2-cell and 4-cell stage embryos that had been cultured from the zygote stage in medium containing small-molecule factors were examined using tetramethylrhodamine methyl ester (TMRM) and 2ʹ,7ʹ-dichlorofluorescein diacetate (DCFDA), respectively. The small-molecule factors reduced mitochondrial activity at both the 2- and 4-cell stages by ≥40% and reduced ROS by >60%. Similarly, staining of 2-cell and 4-cell embryos for GSH levels with tetrafluoroterephthalonitrile indicated that culture of zygotes in the presence of small-molecule factors increased GSH levels. This was supported by data showing an increased GSH/GSSG ratio throughout all stages of preimplantation development, as determined by mass spectrometry. The results are consistent with the ‘quiet embryo’ hypothesis that reduced metabolic activity and ROS favours developmental progression. In summary, these results indicate that small-molecule factors reduce ROS by a combination of suppressing the activity of mitochondria (a major source of cellular ROS) and increasing the concentration of the antioxidant GSH. These results provide insight into how specific factors can be used successfully to improve embryo development in vitro by reducing oxidative stress.