The spatial distribution and functional state of dendritic cells (DCs) are key determinants of tumor immunity. Across complementary studies, we use a high-throughput, longitudinal in vivo platform to visualize tumor growth and immune-cell interactions and define how DC organization shapes disease outcomes. We show that intratumoral cDC1/T-cell clusters serve as local sites of costimulation that promote tumor rejection, whereas spatial separation of these populations is associated with tumor persistence. We further demonstrate that tissue retention of mature DCs can initiate organized, tolerogenic lymphoid niches in homeostasis, while tumor-associated GABA signaling remodels the myeloid microenvironment and contributes to resistance to checkpoint blockade. Together, these findings illustrate how DC positioning and local tissue organization can promote immunity, tolerance, or therapeutic resistance, and identify spatial regulation of DC function as a potential target for improving immunotherapy efficacy.