Fanconi Anemia (FA) results from mutations in a group of genes whose products, including BRCA2 and BACH1/BRIP1, are known to function in one common pathway (the FA-BRCA pathway) to guard genome integrity, especially when challenged by DNA crosslinking agents, such as Cisplatin and mitomycin C (MMC). The extremely high incidence of cancer in FA patients reveals the essentialness of this pathway in tumor suppression. However, this pathway’s involvement in non-FA cancers is not well understood. To evaluate the contribution of the FA-BRCA pathway to cancer, we investigated the integrity of the FA-BRCA pathway in ten human cancer cell lines. We found that the Calu-6 lung cancer cell line carries a defective FA-BRCA pathway. In this cell line, the examination of six FA proteins, essential for the activation of the FA-BRCA pathway, detected substantially reduced expression of FANCL, a catalytic subunit of the ubiquitin ligase/E3-complex. Reconstitution of FANCL in these cells restored the activation of the FA-BRCA pathway, but MMC sensitivity of the cells with a complemented FA-BRCA pathway was decreased as compared to the cells with an impaired FA-BRCA pathway. Collectively, the abnormal FANCL expression is the cause leading to a defective FA-BRCA pathway, which confers the sensitivity of Calu-6 cells to MMC. This suggests that the correlation of an intact FA-BRCA pathway with MMC resistance may emerge as a common mechanism underlying resistance to DNA crosslinking agents in cancer patients.