This article presents the direct isolation of clonogenic human central nervous system stem cells (hCNS-SC) from fresh human fetal brain tissue using antibodies to cell surface markers and fluorescence-activated cell sorting (FACS). These hCNS-SC are phenotypically characterized as SF3 (CD133+), SE12+, CD34+, CD45-, and CD24-1/0. Single CD133+ CD34+ CD45- sorted cells can initiate neurosphere cultures, and their progeny can differentiate into both neurons and glial cells. These cells demonstrate self-renewal potential, as single cells from neurosphere cultures can reestablish neurosphere cultures. Upon transplantation into immunodeficient neonatal mice, the sorted/expanded hCNS-SC show potent engraftment, proliferation, migration, and neural differentiation. The study identifies CD133 as a key marker for isolating these stem cells, and further enrichment is achieved using other markers like SE12 and CD24. The research highlights the potential of hCNS-SC for regenerative medicine and neurological disease treatment. The findings also show that these cells can migrate and differentiate in specific brain regions, responding to host cues without forming tumors. This work provides a foundation for understanding human CNS stem cell biology and their therapeutic applications.This article presents the direct isolation of clonogenic human central nervous system stem cells (hCNS-SC) from fresh human fetal brain tissue using antibodies to cell surface markers and fluorescence-activated cell sorting (FACS). These hCNS-SC are phenotypically characterized as SF3 (CD133+), SE12+, CD34+, CD45-, and CD24-1/0. Single CD133+ CD34+ CD45- sorted cells can initiate neurosphere cultures, and their progeny can differentiate into both neurons and glial cells. These cells demonstrate self-renewal potential, as single cells from neurosphere cultures can reestablish neurosphere cultures. Upon transplantation into immunodeficient neonatal mice, the sorted/expanded hCNS-SC show potent engraftment, proliferation, migration, and neural differentiation. The study identifies CD133 as a key marker for isolating these stem cells, and further enrichment is achieved using other markers like SE12 and CD24. The research highlights the potential of hCNS-SC for regenerative medicine and neurological disease treatment. The findings also show that these cells can migrate and differentiate in specific brain regions, responding to host cues without forming tumors. This work provides a foundation for understanding human CNS stem cell biology and their therapeutic applications.