We aimed to analyze the activation status of commonly deregulated receptor tyrosine kinases (RTK) in human non-small cell lung cancer (NSCLC) tumor initiating cells (TIC) previously demonstrated to be refractory to ionizing radiation and chemotherapy. Phosphorylated RTK and important signaling nodes were assayed using PathScan RTK Signaling Antibody Array Kit in NSCLC TIC and bulk cells 4 h post-irradiation (IR) and validated by Western blot. The effect of mitogen- activated protein kinase kinase (MEK) inhibition combined with IR was analyzed using clonogenic assay. H125 TIC displayed decreased basal phosphorylation of insulin-like growth factor 1 receptor (IGF-1R) and signal transducer and activator of transcription 1 (STAT1) (Tyr701) as compared to bulk cells. Total IGF-1R levels were significantly lower in NSCLC TIC as compared to bulk cells. A higher degree of extracellular signal-regulated kinase (ERK) phosphorylation was evident in TIC and concordantly MEK inhibition reduced TIC viability. Moreover, MEK inhibition also decreased clonogenicity upon IR suggesting that MEK and downstream signaling impart on TIC radiation response. We demonstrate reduced basal phosphorylation of several signaling pathways including lower total IGF-1R levels in NSCLC TIC which is of potential concern for RTK inhibitor use. Importantly, MEK inhibition decreased cell viability of NSCLC TIC alone or in combination with IR.