The hematopoietic system is highly sensitive to radiation exposure, and bone marrow (BM) failure from the hematopoietic acute radiation syndrome (H-ARS) is the primary threat to human life following a nuclear accident or attack. Dimethyl prostaglandin E2 (dmPGE₂) is a promising medical countermeasure (MCM) which significantly increases H-ARS survival in mice. Here mechanisms of dmPGE₂ radiomitigation were defined when given 24 h post-total body irradiation (TBI) in C57BL/6 mice and in humanized NOD scid gamma null (NSG) mice. In C57BL/6 mice exposed to the LD50/30 (8.53 Gy), dmPGE₂ increased cycling rates of hematopoietic stem and progenitor cells (HSPCs) within 6h of treatment, increased total BM cellularity and viability within 24h of treatment, and accelerated peripheral blood recovery. In CD34-humanized NSG mice exposed to 1 Gy, total BM counts were depressed by 48h post-TBI, but % huCD45+ BM chimerism was increased in dmPGE₂-treated animals, suggesting a slight human cell advantage from dmPGE₂. Within the huCD45+ population, significant effects of both TBI and dmPGE₂ on various human HSPC subsets in vivo regarding frequencies, cycling, DNA damage, and gene expression were delineated, including upregulation of transcriptional activators FOS, JUNB, EGR1, and NR4A1 within both mouse and human HSCs in response to dmPGE2 radiomitigation. These proof-of-concept studies support that dmPGE₂ effects related to radiomitigation can be detected in irradiated human HSPCs in vivo in NSG mice. Further work will investigate a more radioresistant humanized mouse system to better model the radiation-damaged BM environment after lethal exposure. Overall, this work describes an innovative translational platform for testing radiation MCMs, and represents a significant step toward human translation of dmPGE₂ for this purpose. The hematopoietic system is highly sensitive to radiation exposure, and bone marrow (BM) failure from the hematopoietic acute radiation syndrome (H-ARS) is the primary threat to human life following a nuclear accident or attack. Dimethyl prostaglandin E2 (dmPGE₂) is a promising medical countermeasure (MCM) which significantly increases H-ARS survival in mice. Here mechanisms of dmPGE₂ radiomitigation were defined when given 24 h post-total body irradiation (TBI) in C57BL/6 mice and in humanized NOD scid gamma null (NSG) mice. In C57BL/6 mice exposed to the LD50/30 (8.53 Gy), dmPGE₂ increased cycling rates of hematopoietic stem and progenitor cells (HSPCs) within 6h of treatment, increased total BM cellularity and viability within 24h of treatment, and accelerated peripheral blood recovery. In CD34-humanized NSG mice exposed to 1 Gy, total BM counts were depressed by 48h post-TBI, but % huCD45+ BM chimerism was increased in dmPGE₂-treated animals, suggesting a slight human cell advantage from dmPGE₂. Within the huCD45+ population, significant effects of both TBI and dmPGE₂ on various human HSPC subsets in vivo regarding frequencies, cycling, DNA damage, and gene expression were delineated, including upregulation of transcriptional activators FOS, JUNB, EGR1, and NR4A1 within both mouse and human HSCs in response to dmPGE2 radiomitigation. These proof-of-concept studies support that dmPGE₂ effects related to radiomitigation can be detected in irradiated human HSPCs in vivo in NSG mice. Further work will investigate a more radioresistant humanized mouse system to better model the radiation-damaged BM environment after lethal exposure. Overall, this work describes an innovative translational platform for testing radiation MCMs, and represents a significant step toward human translation of dmPGE₂ for this purpose.