Colony-stimulating factor-1 (CSF-1) is widely expressed and considered to regulate the development, maintenance, and function of mononuclear phagocyte lineage cells such as monocytes, macrophages, dendritic cells (DCs), Langerhans cells (LCs), microglia, and osteoclasts. Interleukin-34 (IL-34) was recently identified as an alternative ligand for the CSF-1 receptor (CSF-1R) through functional proteomics experiments. It is well established that the phenotype of CSF-1R-deficient (CSF-1R^−/−) mice is more severe than that of mice bearing a spontaneous null mutation in CSF-1 (CSF-1^op/op). CSF-1R^−/− mice are severely depleted of macrophages and completely lack LCs, microglia, and osteoclasts during their lifetime. In contrast, CSF-1^op/op mice exhibit late-onset macrophage development and osteoclastogenesis, whereas they show modestly reduced numbers of microglia and a relatively normal LC development. In contrast, IL-34-deficient (IL-34^−/−) mice show a marked reduction of LCs and a decrease in microglia. IL-34 and CSF-1 display different spatiotemporal expression patterns and have distinct biological functions. In this review, we focus on the functional similarities and differences between IL-34 and CSF-1 in vivo.