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ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense stroma that contributes, along with hypoxia, to immune exclusion and therapeutic resistance. Here, we introduce SpheroMap Cytometry, an innovative spatial flow cytometry platform designed to preserve and quantify the relative localization of immune cells from 3D spheroid models. CAPAN-1 pancreatic tumor cells and either HS-5 bone marrow stromal cells or umbilical cord-derived mesenchymal stromal cells (UC-MSCs) co-cultured in ultra-low-adhesion 96-well plates. PBMC cells (activated or non-activated) were added to infiltrate pre-formed tumor–stromal/mesenchymal spheroids. Then after 48 hours post-seeding and 24 hours post-PBMC incubation, spheroids were incubated with Image-iT Green Hypoxia to mark hypoxic cores, and after 72 hours spheroids were dissociated and stained with CD3, CD4, CD8, CD25 and CD127 antibodies. SpheroMap Cytometry revealed that non-activated immune cells infiltrated spheroids with a distinct pattern between normoxic and hypoxic regions, with an enrichment of CD4 + over CD8 + cells in the hypoxic core and a higher proportion of CD4 + CD25 + CD127 − Treg-like cells. Pre-activation of PBMCs enhanced CD8 + cell infiltration into the hypoxic region and increased CD25 + /CD25 high /CD127 − Tregs in both compartments, indicating that T-cell activation, while facilitating CD8 + cell entry into hypoxic zones, may also promote immunosuppressive populations that impair cytotoxic function. Notably, the effects we observed from the infiltration of pre-activated lymphocytes into tumor–stromal spheroids were not seen in spheroids formed with tumor–mesenchymal cells, which may reflect the immunosuppressive role of umbilical-cord mesenchymal cells. Our findings demonstrate that hypoxia modulates T-cell infiltration and activation, underscoring the critical interplay between oxygen gradients and immune evasion in PDAC. SpheroMap Cytometry provides a scalable, high-resolution method to dissect the spatial immunophenotype of lymphocytes in PDAC models, overcoming limitations of static imaging and conventional flow cytometry, making it an ideal platform for testing therapies aimed at overcoming immune exclusion.