Background: Identifying mechanisms of medulloblastoma recurrence is a key to improving patient survival, and targeting treatment-resistant subpopulations within tumors could reduce disease recurrence. Expression of the granulocyte colony-stimulating factor receptor (G-CSF-R, CD114) is a potential marker of cancer stem cells, and therefore we hypothesized that a subpopulation of medulloblastoma cells would also express CD114 and would demonstrate chemoresistance and responsiveness to G-CSF. Methods: Prevalence of CD114-positive (CD114+) cells in medulloblastoma cell lines, patient-derived xenograft (PDX) tumors, and primary patient tumor samples were assessed by flow cytometry. Growth rates, chemoresistance, and responses to G-CSF of CD114+ and CD114-negative (CD114-) cells were characterized in vitro using continuous live cell imaging and flow cytometry. Gene expression profiles were compared between CD114+ and CD114- medulloblastoma cells using quantitative RT-PCR. Results: CD114+ cells were identifiable in medulloblastoma cell lines, PDX tumors, and primary patient tumors and have slower growth rates than CD114- or mixed populations. G-CSF accelerates the growth of CD114+ cells, and CD114+ cells are more chemoresistant. The CD114+ population is enriched when G-CSF treatment follows chemotherapy. The CD114+ population also has higher expression of the CSF3R, NRP-1, TWIST1, and MYCN genes. Conclusions: Our data demonstrate that a subpopulation of CD114+ medulloblastoma cells exists in cell lines and tumors, which may evade traditional chemotherapy and respond to exogenous G-CSF. These properties invite further investigation into the role of G-CSF in medulloblastoma therapy and methods to specifically target these cells.