Background: The combination of immune checkpoint inhibitors (ICIs) with chemotherapy (chemoimmunotherapy) in the neoadjuvant setting have achieved favorable clinical benefits in non-small cell lung cancer (NSCLC), but the underlying molecular mechanism has not been fully elucidated. Methods: To identify factors associated with the clinical outcome, single-cell RNA/TCR sequencing was performed using 10x Genomics on CD45⁺ immune cells isolated from tumor and multiple immune-relevant tissues and peripheral blood of four treatment-naïve and eight neoadjuvant chemoimmunotherapy treated IIIA NSCLC patients (responders versus non-responders). Bioinformatics analysis was used to screen immune cell factors associated with therapy response in single-cell sequencing profiles, and results were verified at protein level by flow cytometry, multiplex fluorescent immunohistochemistry (mIHC) and ELISA. Results: A total of 186,477 immune cells from 48 samples were acquired after quality-control filtering. The synergistic increase of B cells and CD4⁺ T cells are associated with positive therapeutic response of neoadjuvant chemoimmunotherapy. B cell class switching to IgG1 and IgG3 plays a critical role in anti-tumor immune response in tumor lesion, and this process is driven by increased IL-21 protein secreted by infiltrated T follicular helper (Tfh) cells after neoadjuvant chemoimmunotherapy. Several critical events lead to the positive clinical outcome during neoadjuvant chemoimmunotherapy, including the diminished activated TNFRSF4⁺ regulatory T cells (Tregs), increased LAMP3⁺ dendritic cells (DCs), high pretreatment peripheral blood T-cell diversity, and the expansion of intratumoral CD4⁺ T clones and peripheral CD8⁺ T clones. A validation cohort of 26 treatment-naïve and 30 neoadjuvant chemoimmunotherapy treated IIIA NSCLC patients verified these findings. Conclusions: Single-cell profiling of the immune response to neoadjuvant chemoimmunotherapy uncovered several critical factors of anti-tumor immune response and provided insight of potential novel predictive factors and therapeutic targets for improving the efficacy of treatment in NSCLC.