Neuroinflammation is a characteristic of almost all neurological diseases. Since microglia, the resident macrophages of the central nervous system (CNS), are key players in neuroinflammatory processes there is an increasing interest in this cell type as a therapeutical target to suppress neuroinflammation. Thorough biological knowledge of microglia is therefore of pivotal importance, and microglia in vitro models (in a culture dish) are excellent means to obtain such knowledge. However, there is currently no in vitro microglia model that recapitulates all the characteristic features of in vivo microglia (in a living organism), which hampers the in vitro-in vivo translation. For instance, microglia in the healthy CNS are characterized by a ramified morphology, whereas in vitro microglia are characterized by a more amoeboid (rounder) morphology. In addition, the gene expression profile of in vitro microglia differs considerably from the gene expression profile of ex vivo microglia (freshly isolated microglia). Several studies have reported that cues from the CNS environment are important for microglia to establish or maintain their identity. However, which CNS environmental cues contribute to the in vivo microglia gene expression profile that defines their identity is poorly understood. In this thesis, we used a transcriptomic-guided approach to uncover cues that shape microglia identity and investigated if they could be used to optimize in vitro culture conditions to study microglia in health and disease. The results in this thesis have led to the development of a partly serum-free culture protocol that yields high numbers of ramified microglia with a transcriptome profile similar to that of microglia that were cultured in serum-free medium only. Additional transcriptome and in silico (computational) analyses throughout this thesis have provided powerful leads to further improve microglia in vitro culture conditions, for instance through i) the addition of intercellular microglia signaling cues, ii) the exposure to extracellular matrix components and iii) the generation of more biophysically accurate culture methods. Better microglia in vitro models are pivotal to further understand the biology of microglia, and are also important to reduce and replace animal testing.