Abstract Both cell-intrinsic and microenvironmental influences vitally govern tumor progression. Mutational, chromosomal and epigenetic alterations represent the former, while the latter is influenced by diverse stromal populations including fibroblasts, vasculature and immune cells. In vitro organoid culture provides an experimentally tractable platform for cancer research that is applicable to cell-autonomous and tumor microenvironmental studies. The first part of this lecture will describe our work using organoid culture to functionally validate tumor cell-intrinsic genomic alterations. Notably, cancer research has traditionally lacked in vitro methods where tumors can be holistically cultured in a non-reconstitutive manner that co-preserves neoplastic cells together with endogenous stromal components. Thus, the second part of this lecture will describe advances in 3D tumor organoid culture that allow growth of cancer cells in a manner that preserves their native interaction with diverse tumor-infiltrating immune cells and allows analysis of immune checkpoint inhibition. Citation Format: C. Kuo. Organoid Models of Cancer and the Tumor Microenvironment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr Wrk2-01.