Integrins play crucial roles in epithelial adhesion, proliferation, wound healing and cancer. In the epidermis, the roles of many integrin subunits are incompletely defined and mechanistic details regarding their functions are lacking. We performed a multiplexed shRNA screen to define roles for each subunit in human organotypic skin. This screen identified the integrin αv class of heterodimers as essential for generation of human skin tissue. We demonstrate that integrin αv loss drives a keratinocyte G1-S cell cycle checkpoint block. Surprisingly, αv integrins are not localized within keratinocyte focal adhesions and instead maintain proliferation by controlling c-myc translation through FAK, p38 and p90RSK signaling pathways. These phenotypes depend only on αv’s binding partners β5 and β6, but not β1 or β8. Utilizing inducible genetic depletion of integrin αv, or blocking antibodies targeting αv heterodimers, we show that αv integrins are required for de novo tissue generation, but dispensable for epidermal maintenance. In an in vivo human xenograft skin model, we use blocking antibodies to show that integrin αv is required for epidermal proliferation during wound healing, but is dispensable for normal epidermal homeostasis. In organotypic human neoplasias driven by Cdk4 R24C and oncogenic H-Ras G12V, we show that integrin αv is necessary for neoplastic tissue thickness and invasion through the basement membrane. This is dependent on expression of both binding partners β5 and β6. Blocking antibodies targeting αv heterodimers reduce tumor burden and proliferation in an inducible, orthotopic xenograft cutaneous squamous cell carcinoma tumor model. In conclusion, we demonstrate, for the first time, essential roles for αv integrins in human cutaneous wound re-epithelialization and tumorigenesis. We further determine a novel focal adhesion-independent signaling mechanism for αv’s involvement in cell cycle progression.