© 2017, Springer Science+Business Media New York. Protein kinase CK2 is a ubiquitously expressed serine/threonine kinase composed of two catalytic subunits (α) and/or (α’) and two regulatory (β) subunits. The expression and kinase activity of CK2 is elevated in many different cancers, including glioblastoma (GBM). Brain tumor initiating cells (BTICs) are a subset of cells that are highly tumorigenic and promote the resistance of GBM to current therapies. We previously reported that CK2 activity promotes prosurvival signaling in GBM. In this study, the role of CK2 signaling in BTIC function was examined. We found that expression of CK2α was increased in CD133+ BTICs compared to CD133− cells within the same GBM xenolines. Treatment with CX-4945, an ATP-competitive inhibitor of CK2, led to reduced expression of Sox2 and Nestin, transcription factors important for the maintenance of stem cells. Similarly, inhibition of CK2 also reduced the frequency of CD133+ BTICs over the course of 7 days, indicating a role for CK2 in BTIC persistence and survival. Importantly, using an in vitro limiting dilution assay, we found that inhibition of CK2 kinase activity with CX-4945 or siRNA knockdown of the CK2 catalytic subunits reduced neurosphere formation in GBM xenolines of different molecular subtypes. Lastly, we found that inhibition of CK2 led to decreased EGFR levels in some xenolines, and combination treatment with CX-4945 and Gefitinib to inhibit CK2 and EGFR, respectively, provided optimal inhibition of viability of cells. Therefore, due to the integration of CK2 in multiple signaling pathways important for BTIC survival, CK2 is a promising target in GBM.