Neuroblastoma (NB) is an often fatal pediatric tumor of neural crest origin. We previously isolated NB tumorinitiating cells (NB TIC) from bone marrow metastases that resemble cancer stem cells and form metastatic NB in immunodeficient animals with as few as ten cells. To identify signaling pathways important for the survival and self-renewal of NB TICs and potential therapeutic targets, we screened a small molecule library of 143 protein kinase inhibitors, including 33 in clinical trials. Cytostatic or cytotoxic drugs were identified that targeted PI3K (phosphoinositide 3-kinase)/Akt, PKC (protein kinase C), Aurora, ErbB2, Trk, and Polo-like kinase 1 (PLK1). Treatment with PLK1 siRNA or low nanomolar concentrations of BI 2536 or BI 6727, PLK1 inhibitors in clinical trials for adult malignancies, were cytotoxic to TICs whereas only micromolar concentrations of the inhibitors were cytotoxic for normal pediatric neural stem cells. Furthermore, BI 2536 significantly inhibited TIC tumor growth in a therapeutic xenograft model, both as a single agent and in combination with irinotecan, an active agent for relapsed NB. Our findings identify candidate kinases that regulate TIC growth and survival and suggest that PLK1 inhibitors are an attractive candidate therapy for metastatic NB. Cancer Res; 71(4); 1385-95. Ó2011 AACR.