We developed a novel humanized B-hCD3EDG/h4-1BB mouse model by crossing mice expressing chimeric CD3E, CD3D, and CD3G with those expressing the human extracellular region of 4-1BB. Flow cytometry confirmed expression of human CD3 and 4-1BB on splenocytes in homozygous mice, with no murine CD3 or 4-1BB expression. Assessment of leukocyte subpopulations revealed no significant alterations, indicating that humanization did not affect blood cell populations or the CD4/CD8 T cell ratio in various tissues. This model provides a robust platform for evaluating novel T cell engagers.