Mast cells (MCs) are long-lived immune cells, which were reported to play an important role in initiating innate and adaptive immune responses against various infections. MCs accumulate in high numbers in the stroma and at the invasion front of various human cancers, suggesting a possible contribution by MCs to tumour growth. Experimental studies using crosses of MC-deficient Kit-mutant mouse strains with mouse models of epithelial cancers have provided evidence for important MC tumour-promoting functions. However, the complex alterations of the immune system that characterize Kit-mutant mice in addition to their MC deficiency, limit the interpretation of these findings. Numerous key observations made in Kit mutant mice were not reproduced in novel, Kit-independent mouse models of MC deficiency. Thus, the impact of MCs on tumour biology remains unclear. The aim of this study is to clarify the contribution of MCs to the biology of Human papilloma virus (HPV)-induced skin cancer in a Kit-independent mouse model of MC deficiency. In K14-HPV16 transgenic mice, HPV oncogenes are constitutively expressed in the epidermis resulting in epidermal hyperplasia with 100% penetrance and squamous cell carcinoma in about 50% of the animals. A cross to a Kit-mutant line suggested that MCs are important tumour promoters in this model. We crossed K14-HPV16 mice to M5Cre R-DTA line, in which MCs are constitutively depleted with high efficiency and selectivity. Unexpectedly, the loss of MCs neither affected keratinocyte proliferation indices nor altered keratinocyte apoptosis at any stage of HPV-induced neoplasia. Furthermore, the loss of MCs did not result in any detectable changes in composition and gene expression of the inflammatory hematopoietic cell infiltrate in the tumour stroma. This shows that, contrary to current belief, MCs have no important function in orchestrating the tumour micro milieu. In keeping with this finding, MC deficiency resulted in no detectable difference in the incidence growth or grading of SSC in K14-HPV16 transgenic mice. Collectively, these results show that, despite their high density in HPV-induced neoplasia, MC have no role in cancerogenesis or neoplastic progression in the K14-HPV16 mouse model. Our findings also emphasize the importance of novel Kitindependent mouse models in the investigation of MC in vivo functions.