In order to predict efficacy and toxicity of compounds advancing in the oncology drug discovery pipeline, and to minimize requirements for multiple animal studies, the development of in vitro assays that recapitulate in vivo conditions is increasingly important. While high throughput screening favors 2D assays, both for cost and relative speed in culturing and plating, they do not recapitulate the tumor architecture and the microenvironment that is more comparable to in vivo models. We now report this has been accomplished using zPREDICTA's organ-specific three-dimensional (3D) culture models which support long-term culture of carcinoma cells. Physiologic extracellular matrix (ECM) components are critical in maintaining the physical and spatial microenvironment as well as tumor phenotypes in reconstructed bone marrow (r-Bone) and mouse breast (r-mBreast) models. The r-Bone ECM allows for co-culture of tumor cells with mesenchymal stem cells, fibroblasts, lymphoid, myeloid, and CAR-T cells. The data here describes clear differences in the in vitro pharmacology of several standard of care agents in 2D, in adherent or suspension cultures, compared to the recapitulated 3D tumor microenvironment of the human hematopoietic compartment or the mouse breast ductal epithelium. For example, we find that cisplatin has a 10-30 fold higher IC50 in 4 different murine cell lines (MMTV-PyMT, 4T1, EMT6, and E0771) when cultured in the r-mBreast model than when those cells are adhered to the polystyrene in a 2D culture. Cytotoxicity assays such as CellTiter-Glo® can also be performed in a high throughput format in both 96 and 384-well 3D assay formats and we demonstrate equivalent bortezomib IC50 values in RPMI 8226 cell r-Bone cultures in both plate types. Additionally, cells can be isolated from the matrix after test agent exposure and analyzed by an appropriate downstream application, such as flow cytometry, where we show anti-CD19 CAR-T cell killing of CD19+ NALM6 cells in r-Bone cultures. These 3D models represent new early opportunities to screen test agents in a platform that is designed to more closely mimic in vivo tissue microenvironments and serve as a more relevant tool for multi drug screening and as a prerequisite to animal studies. Citation Format: Michael E. Steffey, Ying Qu, Andrew Karalewitz, Sumithra Urs, Julia Kirshner, Scott Wise. Human and mouse reconstructed plate-based 3-dimensional culture assays that mimic the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2643.