Background: Immunotherapies acting upon tumor infiltrating lymphocytes (TILs) generate deep and durable responses in NSCLC patients. However, the clonality, cell phenotype, and signaling states of TILs remain poorly defined in clinical specimens. In addition, current in vitro model systems do not typically preserve both the tumor epithelium and microenvironment as an intact syngeneic unit thus hindering conceptual and therapeutic advances. Methods: We developed 3-dimensional organoid cultures of surgically resected NSCLC specimens that intrinsically retained diverse tumor microenvironmental cellular components without requiring reconstitution. We then profiled TIL clonotypes and gene expression phenotypes in varying histologies of NSCLC with a 5’ based scRNA-seq platform capable of pairing alpha beta T cell receptor (TCR) sequence identity with 5’ based transcriptomes. Results: Fresh clinical samples were viably dissociated into single cell suspensions and droplet based scRNA-seq was carried out in a rapid and reliable manner. Correct, full length, and paired TCRA/TCRB clonotypes were detected at expected ratios and demonstrated a detection sensitivity of clonal expansion with a sensitivity of 1% for 1000 cells observed. In addition, paired transcriptome analysis facilitated phenotypic categorization of each unique clonotype, including cytotoxic and helper T, B, Treg, as well as TIL exhaustion. Furthermore, examination of NSCLC tumor microenvironments with varying patient smoking history and tumor histology exhibited differences suggesting a potential link between immune microenvironments and cancer etiology. Conclusions: Organoid cultures faithfully recapitulate tumor microenvironment diversity within NSCLC histologic types, and scRNA-seq profiling of clonotype TIL dynamics may elucidate underlying immune microenvironment dynamics in NSCLC patients. Further studies in this human preclinical model coupled with scRNA-seq may advance our understanding of tumor immunology within NSCLC subtypes as well as facilitate the discovery of novel immunotherapies.