Langerhans cells (LCs) are the exclusive antigen-presenting cells of the epidermis, capable of mounting immunity and tolerance. LCs maintain themselves locally by self-renewing throughout life, a process that is regulated by both LCs and keratinocytes. Nevertheless, the mechanisms underlying this process are not clearly understood. Using targeted genetic ablation, we demonstrate that lack of protein S (PROS1) in keratinocytes, but not in LCs, results in reduced numbers of terminally developed LCs. This is due to increased apoptosis of LCs and associated with altered expression of cytokines involved in tissue homeostasis. Furthermore, PROS1 also down-regulates LC differentiation from bone marrow precursors. Together, these identify PROS1 as a regulator of LC development and homeostasis.