EIF2AK3 encodes PKR-like endoplasmic reticulum (ER) kinase (PERK), which regulates ER stress response and plays a crucial role in deciding the cell’s commitment to survival/adaptation, stress tolerance, or apoptosis, depending on stress severity, duration, and recurrence. Conversely, common single nucleotide variants (SNVs) of EIF2AK3 may be associated with an increased risk of disorders in both the periphery and the central nervous system, albeit with small-medium effect sizes. Three exonic EIF2AK3 SNVs are in linkage disequilibrium and form the PERK-B haplotype, which occurs in 28% of the global population, compared to PERK-A, which is present in 62% of the population. The PERK-B haplotype was reported to be associated with several chronic neurodegenerative diseases, including progressive supranuclear palsy, neurocognitive impairment in people living with HIV, and Alzheimer’s. Importantly, the precise impact of these SNVs on PERK activity and function remains elusive. We have developed novel triple knock-in mice expressing the three exonic PERK-B SNVs. When exposed to acute ER stress, PERK-B mice exhibited elevated PERK kinase activity in the liver and heightened vulnerability to apoptotic cell death in the pancreas, compared to PERK-A mice. Furthermore, we used primary neuroglial cells from the PERK-B or PERK-A mice to examine cell survival, stress response, and stress tolerance. Thapsigargin-induced stress response of neuroglial cultures demonstrates differential and transient changes with PERK-B compared to PERK-A. PERK-B neuroglial cultures, but not enriched neurons, exhibited higher levels of eIF2 phosphorylation at 4-6 h, and activation of PERK signaling, specifically proapoptotic CHOP, at 12 h. However, ER stress-mediated death at 24 h was comparable between the PERK-A and PERK-B neuroglia. We also observed differential stress tolerance between the PERK-A and PERK-B neuroglial cultures, wherein PERK-B neuroglial cultures pretreated with a low-dose PERK-specific activator exhibited increased vulnerability to NMDA -induced death compared to PERK-A cultures. In summary, PERK-B was associated with transient changes in stress response and decreased stress tolerance with higher vulnerability to cellular stress. These changes, albeit transient and small/moderate sized, have clinical relevance as they might contribute to the overall genetic vulnerability in chronic diseases.