The aim of this study is to establish an in vitro co-culture system to model allograft rejection using kidney organoids system derived from human induced pluripotent stem cells (hiPSCs). We co-cultured kidney organoids derived from wild-type hiPSCs with HLA-mismatched peripheral blood mononuclear cells (PBMCs) from healthy controls (HC) for 24 hours. To assess allogeneic rejection modeling, we measured the expression of HLA molecules, (HLA-ABC and HLA-DR), and evaluated cellular damage in the kidney organoids. Additionally, we analyzed the distribution of T cells and their subsets within the co-cultured PBMCs. The immunosuppressive effect of tacrolimus was also evaluated in this co-culture system. Transcriptomic analysis, conducted using RNA sequencing, identified molecules associated with allogeneic rejection. When kidney organoids were co-cultured with alloreactive PBMCs for 24 hours, HLA-ABC and HLA-DR expression significantly increased in kidney organoid cells. Additionally, kidney organoids showed reduced cell viability and increased apoptosis compared to syngeneic controls, as assessed by flow cytometry and Annexin V/PI staining. However, treatment with tacrolimus reduced HLA expression in a dose-dependent manner, highlighting the diminished alloimmune responses. Further analysis of PBMC subsets revealed shifts in T helper (TH) and cytotoxic T cell (TC) populations under allogeneic conditions, including increased effector TH and TC cells. Transcriptomic analysis through RNA sequencing identified 256 differentially expressed genes (DEGs), with notable immune-related pathways such as NF-kappa B and TNF signaling involved in allograft rejection. These results provide evidence that a co-culture system with allogeneic kidney organoids and PBMCs can potentially model transplant rejection in vitro.