Microglia are the resident macrophages of the central nervous system (CNS) and play a key role in CNS development, homeostasis, and disease. Goodin vitromodels are indispensable to study their cellular biology, and although much progress has been made,in vitrocultures of primary microglia still only partially recapitulate the transcriptome ofin vivomicroglia. In this study, we explored a combination ofin silicoandin vitromethodologies to gain insight into cues that are involved in the induction or maintenance of theex vivomicroglia reference transcriptome. First, we used thein silicotool NicheNet to investigate which (CNS-derived) cues could underlie the differences between the transcriptomes ofex vivoandin vitromicroglia. Modeling on basis of gene products that were found to be upregulatedin vitro, predicted that high mobility group box 2 (HMGB2)- and interleukin (IL)-1β-associated signaling pathways were driving their expression. Modeling on basis of gene products that were found to be downregulatedin vitro, did not lead to predictions on the involvement of specific signaling pathways. This is consistent with the idea thatin vivomicroenvironmental cues that determine microglial identity are for most part of inhibitory nature. In a second approach, primary microglia were exposed to conditioned medium from different CNS cell types. Conditioned medium from spheres composed of microglia, oligodendrocytes, and radial glia, increased the mRNA expression levels of the microglia signature geneP2RY12.NicheNet analyses of ligands expressed by oligodendrocytes and radial glia predicted transforming growth factor beta 3 (TGF-β3) and LAMA2 as drivers of microglia signature gene expression. In a third approach, we exposed microglia to TGF-β3 and laminin.In vitroexposure to TGF-β3 increased the mRNA expression levels of the microglia signature geneTREM2. Microglia cultured on laminin-coated substrates were characterized by reduced mRNA expression levels of extracellular matrix-associated genesMMP3andMMP7, and by increased mRNA expression levels of the microglia signature genesGPR34andP2RY13. Together, our results suggest to explore inhibition of HMGB2- and IL-1β-associated pathways inin vitromicroglia. In addition, exposure to TGF-β3 and cultivation on laminin-coated substrates are suggested as potential improvements to currentin vitromicroglia culture protocols.