Increased awareness of pathogens with pandemic potential, especially respiratory viruses, is driving research on next-generation mucosal vaccines. However, clinical translation is still hampered by the lack of relevant experimental systems. Here, we review advances in human mucosal ex vivo cultures and their eligibility for vaccine development as an alternative to animal models. Ranging from organotypic air-liquid interface cultures to lymphoid organoids and microfluidics-based co-cultures, several breakthroughs occurred in recent years in modeling mucosa architecture and physiology, as well as adaptive immune responses. Advancing recent progress for clinical developments may require high-throughput approaches to validate the representativeness of the immune response within models, benchmark best practices for regulatory standardization, and investigate the influence of microbiota on mucosal immune responses.