Viral and bacterial lung infections place a significant burden on public health. Versican, an extracellular matrix (ECM) chondroitin sulfate proteoglycan, is a critical coordinator of the innate immune response. Versicans potential as an immunomodulatory molecule makes it a promising therapeutic target for controlling the host’s immune response to lung infection. However, versicans contribution to lung inflammation, injury, and immune cell activity during influenza A virus (IAV) infection represents a critical knowledge gap. In the following work, we characterized the spatiotemporal accumulation of versican in the lungs of wild-type (C57BL/6J, WT) mice in response to IAV and found that versican accumulation correlates with mouse acute lung injury scores and pulmonary inflammatory cell infiltration. We found that versican expression is increased in both mononuclear phagocytic cells and stromal cells in the lungs in response to IAV in WT mice and discovered that the expression of versican in stromal cells is partially mediated by type I interferon receptor signaling. These data from WT mice raised intriguing questions about the role of versican in the early host response to IAV. To rigorously investigate the potential of versican to modulate lung inflammation, we developed a complete protocol combining spectral flow cytometry with in vivo compartmental analysis, for the precise localization of multiple immune cell populations participating in the pulmonary host response to IAV. The lungs have high autofluorescence (AF) compared to other tissues, which complicates the analysis of pulmonary leukocytes by flow cytometry. In the following work, we described changes in the AF characteristics of control and IAV-infected lungs during the transition from innate to adaptive immunity. We meticulously assembled a broad panel of 20 antibody-fluorophore conjugates compatible with these AF characteristics and spectral flow cytometry. Additionally, our studies validated that incorporating multiple heterogeneous AF signatures from the lungs improves the resolution and identification of fluorescence signals, particularly when alveolar macrophages are a component of the immune response. The methodology presented includes a robust gating strategy for the identification of B cells, T cells (cytotoxic T, T helper, and gd T cells), NK cells, macrophages (alveolar, recruited and monocyte-macrophages), monocytes (Ly6Clo and Ly6Chi), dendritic cells (CD103+ and CD11b+), neutrophils, and eosinophils in combination with dual in vivo CD45 labeling to facilitate identification of these immune cell populations in four pulmonary compartments. Finally, our studies implemented informed gating and dimensionality reduction algorithms to visualize the recruitment and migration of leukocytes from the vasculature, across the lung interstitium, and into the alveolar airways at 9 days post-infection with IAV. Our robust spectral flow cytometry panel and in vivo intravascular CD45 labeling technique were applied to studies investigating the role of versican in lung inflammation, injury, and immune cell activity during IAV infection. To address our central hypothesis that fibroblastderived versican is pro-inflammatory and enhances the innate immune response to IAV infection, we generated a tamoxifen-inducible mouse strain that is deficient in fibroblast-derived versican (B6. Col1a2-CreERT+/- /Vcantm1.1Cwf, Col1a2/Vcan-/- ). We reported that fibroblast-derived versican plays a critical role in neutrophil, monocyte, mono-macrophage, dendritic cell, and eosinophil migration into the lungs and airways early in IAV infection. Intriguingly, fibroblastderived versican deficiency had the most substantial impact on neutrophil emigration into the lungs. We found that Col1a2/Vcan-/- mouse lung fibroblasts (mLFs) have reduced cell-associated hyaluronan (HA) and that neutrophils were less adhesive to the versican/HA ECM of Col1a2/Vcan-/- mLFs compared to WT controls. These findings suggest that fibroblast-derived versican and associated HA are necessary for the adhesion of neutrophils to the lung fibroblasts as they transit into the lung interstitium and airways from the pulmonary vasculature. Our findings demonstrate that fibroblast-derived versican is a key integrator of the early host immune response to IAV.