The immune system consists of specialized cell types with distinct functions in order to provide an effective innate and adaptive immune defense against harmful invading pathogens like bacteria, viruses, fungi, parasites, or other substances threatening the integrity of the organism. Once the immune system recognizes such pathogens via pattern recognition receptors (PRRs), they are taken up, processed, and presented as antigens on MHC class I and II to T lymphocytes by specialized cells called dendritic cells (DCs). At the same time pathogen components which bind to PRRs in DCs trigger potent cytokine and chemokine responses. Although other cell types like macrophages can also take up, process, and present antigens to naïve T lymphocytes, DCs are the cells with the greatest capacity to do so. Thus, DCs are also called professional antigen presenting cells (APCs), which induce a strong adaptive immune response and thereby act as a bridge between the innate and adaptive immune system. This chapter provides detailed instructions on how to generate various types of DCs from human peripheral blood mononuclear cells (PBMCs) and murine bone marrow, as well as stimulation conditions for activation of these cells by PRR ligands in vitro.