Recent studies highlighted the importance of astrocytes in neuroinflammatory diseases, interacting closely with other CNS cells but also with the immune system. However, due to the difficulty in obtaining human astrocytes, their role in these pathologies is still poorly characterized. Here, we develop a serum-free protocol to differentiate human induced pluripotent stem cells (hiPSCs) into astrocytes. Gene expression and functional assays show that our protocol consistently yields a highly enriched population of resting mature astrocytes across the 13 hiPSC lines differentiated. Using this model, we first highlight the importance of serum-free media for astrocyte culture to generate resting astrocytes. Second, we assess the astrocytic response to IL-1β, TNF-α, and IL-6, all cytokines important in neuroinflammation, such as multiple sclerosis. Our study reveals very specific profiles of reactive astrocytes depending on the triggering stimulus. This model provides ideal conditions for in-depth and unbiased characterization of astrocyte reactivity in neuroinflammatory conditions. Display omitted •Human iPSCs are differentiated from blood of control and multiple sclerosis subjects•Functional astrocytes are differentiated from hiPSC in serum-free conditions•In-depth characterization of the effect of serum on astrocyte phenotype•Multiple sclerosis cytokines trigger specific activation profiles in astrocytes Perriot et al. describe a new protocol to differentiate both resting and reactive astrocytes from human induced pluripotent stem cells in serum-free conditions. They show that astrocytes exhibit highly contrasted responses to serum and to cytokines involved in multiple sclerosis, establishing that this model is suitable to study neuroinflammation.