The treatment of non-small cell lung cancer has made major strides with the use of immune checkpoint inhibitors; however, there remains a significant need for therapies that can overcome immunotherapy resistance. Dendritic cell (DC) vaccines have been proposed as a therapy that can potentially enhance the antitumor immune response. We have embarked on a phase I clinical trial of a vaccine consisting of monocyte-derived DCs (moDC) modified to express the chemokine C-C motif chemokine ligand 21 (CCL21-DC) given in combination with pembrolizumab. In this study, we report a comprehensive characterization of this CCL21-DC vaccine and interrogate the effects of multiple factors in the manufacturing process. We show that the cellular makeup of the CCL21-DC vaccine is heterogeneous because of the presence of passenger lymphocytes at a proportion that is highly variable among patients. Single-cell RNA sequencing of vaccines revealed further heterogeneity within the moDC compartment, with cells spanning a spectrum of DC phenotypes. Transduction with a CCL21-containing adenoviral vector augmented CCL21 secretion by moDCs, but otherwise had a minimal effect on vaccine characteristics. A single freeze-thaw cycle for stored vaccines was associated with minor alterations to the DC phenotype, as was the use of healthy donors rather than patient autologous blood. Our results highlight important considerations for the production of DC vaccines and identify underexplored factors that may affect their efficacy and immunologic impact.