BACKGROUND: Transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2) plays a key role in detoxification of electrophiles via formation of glutathione (GSH) adducts and subsequent excretion into extracellular spaces. We found that reactive sulfur species (RSS), such as cysteine persulfides produced by cystathionine c-lyase (CSE), capture environmental electrophiles through formation of sulfur adducts. However, contributions of Nrf2 and CSE to the blockage of environmental electrophile-mediated toxicity remain to be evaluated. OBJECTIVES: The aim of this study was to clarify roles that CSE and Nrf2 play in the protection against various environmental electrophiles. We also wished to clarify the molecular basis of the developmental window of toxicity through investigating expression levels of Nrf2, RSS-producing enzymes, and sulfur nucleophiles during developmental stages of mice. METHODS: Wild-type (WT), CSE knockout (KO), Nrf2 KO, Nrf2/CSE double KO (DKO) mice, and their primary hepatocytes were analyzed in this study. Cadmium (Cd), methylmercury (MeHg), 1,4-naphthoquinone, crotonaldehyde, and acrylamide were used. We conducted Western blotting, real-time polymerase chain reaction (PCR), 3-(4,5-dimethylthiazol-2-yl)-2,5-triphenyl tetrazolium bromide (MTT) assays, liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) analysis, alanine transaminase (ALT) activity, histopathological analysis, and rotarod test. RESULTS: Primary hepatocytes from DKO mice were significantly more sensitive to the environmental electrophiles than each single KO counterpart. Both Nrf2 and CSE single KO mice were highly susceptible to Cd and MeHg, and such sensitivity was further exacerbated in the DKO mice. Lower-level expressions of CSE and sulfur nucleophiles than those in adult mice were observed in a window of developmental stage. CONCLUSIONS: Our mouse model provided new insights into the response to environmental electrophiles; while Nrf2 is recognized as a key transcription factor for detoxification of environmental electrophiles, CSE is crucial factor to repress their toxicity in a parallel mode. In addition, the sensitivity of fetuses to MeHg appears to be, at least in part, associated with the restricted production of RSS due to low-level expression of CSE. https://doi.org/10.1289/EHP4949.