While several studies have presented evidence that both mouse and human adult cortical astrocytes can dedifferentiate and form multipotent neurospheres after brain injury, other observations support that these neurospheres are formed by astroglia progenitors derived from subependymal zone (SEZ) resident neural stem cells which migrated to the cortex. In this study, we developed a new gliosis culture model composed by cells from the injured cortex, including reactive astrocytes, microglia, macrophages, and NG2 cells that mimic some characteristics of the injured brain but in a more controlled lab setting, allowing us to better explore the origin of the neurospheres mentioned above. When non-reactive adult glial cells from either the adult mouse cortex or the substantia nigra were cocultured with these reactive cells, the former generated multipotent neurospheres. Considering that the SEZ was not included in the cortical and SN dissected areas, our results support that adult glial cells are indeed capable of de-differentiating after being exposed to injury. Next, we used different culture models and proteomic analyses to reveal that myeloid cells are involved in and can trigger the dedifferentiation of cortical astroglia by secreting certain molecules. Indeed, bone-marrow-derived myeloid cells placed in transwells, which permit transmission of molecular signals but not cells, also led to the formation of neurospheres from adult glial cells co-cultured in adjacent chambers. These results demonstrate that myeloid cells, which significantly increase in numbers after acute tissue damage through infiltration or proliferation, can truly induce fate plasticity of adult postmitotic glial cells in the injured brain.