Background: Rare diseases, which affect millions of people worldwide, pose a major challenge, as it often takes years before an accurate diagnosis can be made. This delay results in substantial burdens for patients and health care systems, as misdiagnoses lead to inadequate treatment and increased costs. Artificial intelligence (AI)–powered symptom checkers (SCs) present an opportunity to flag rare diseases earlier in the diagnostic work-up. However, these tools are primarily based on published literature, which often contains incomplete data on rare diseases, resulting in compromised diagnostic accuracy. Integrating expert interview insights into SC models may enhance their performance, ensuring that rare diseases are considered sooner and diagnosed more accurately. Objective: The objectives of our study were to incorporate expert interview vignettes into AI-powered SCs, in addition to a traditional literature review, and to evaluate whether this novel approach improves diagnostic accuracy and user satisfaction for rare diseases, focusing on Fabry disease. Methods: This mixed methods prospective pilot study was conducted at Hannover Medical School, Germany. In the first phase, guided interviews were conducted with medical experts specialized in Fabry disease to create clinical vignettes that enriched the AI SC’s Fabry disease model. In the second phase, adult patients with a confirmed diagnosis of Fabry disease used both the original and optimized SC versions in a randomized order. The versions, containing either the original or the optimized Fabry disease model, were evaluated based on diagnostic accuracy and user satisfaction, which were assessed through questionnaires. Results: Three medical experts with extensive experience in lysosomal storage disorder Fabry disease contributed to the creation of 5 clinical vignettes, which were integrated into the AI-powered SC. The study compared the original and optimized SC versions in 6 patients with Fabry disease. The optimized version improved diagnostic accuracy, with Fabry disease identified as the top suggestion in 33% (2/6) of cases, compared to 17% (1/6) with the original model. Additionally, overall user satisfaction was higher for the optimized version, with participants rating it more favorably in terms of symptom coverage and completeness. Conclusions: This study demonstrates that integrating expert-derived clinical vignettes into AI-powered SCs can improve diagnostic accuracy and user satisfaction, particularly for rare diseases. The optimized SC version, which incorporated these vignettes, showed improved performance in identifying Fabry disease as a top diagnostic suggestion and received higher user satisfaction ratings compared to the original version. To fully realize the potential of this approach, it is crucial to include vignettes representing atypical presentations and to conduct larger-scale studies to validate these findings.