The maintenance and repair of the myocardium following diffuse injury includes a critical contribution from the endogenous cardiac stem cell population (Ellison et al., 2013). The development of receptor tyrosine kinase inhibitors as anti-cancer therapies has advanced the treatment of cancer, but has also been associated with cardiotoxic side-effects (Albini et al., 2010). Although a principal component of cardiotoxicity is damage to cardiomyocytes, impact on the stem cell population could play a key role in the long-term failure of the heart to recover from this injury. We have therefore here investigated the impact of three receptor tyrosine kinase inhibitors (imatinib mesylate; sunitinib maleate; sorafenib tosylate), examining potential pathways of cell death at clinically comparable concentrations. To study these effects on the endogenous cardiac stem cell population specifically, we have used human cardiac stem cells, isolated by magnetic cell sorting to obtain c-kit-positive, CD45-negative cells (Smith et al., 2014). Cells were characterised and maintained in vitro, applying the three selected drugs at concentrations equivalent to peak and trough clinical plasma levels. Impact on cardiac stem cell viability and activation of cell death pathway mechanisms were first assessed by fluorescein diacetate viability assay (Smith et al., 2009) and live cell staining to identify apoptotic or necrotic pathway activation. Further real-time qPCR analysis of apoptosis- and necrosis-related genes, TUNEL staining and immunocytochemistry provided a broad screen of underlying mechanisms. All three drugs caused reduced cell viability (10µM imatinib for 24 hours: 70±11%; 10 µM sorafenib for 24 hours: 67±8%; 2 µM sunitinib for 24 hours: 74±6%; relative to untreated control values of 100+6.5%, n=7, p<0.05, mean±s.e.m., Tukey’s). Examination of caspase-3/7 activation showed no increase following sorafenib or imatinib treatment, however sunitinib (20 µM) caused a 23±2.5% increase in caspase-3/7-positive cells (n=4, p<0.05, Tukey’s). Real-time qPCR analysis showed increased expression of genes linked with apoptosis after sunitinib (2 µM) exposure, with 3±0.7 fold-change in calpain, 2.5±1.4 in FASR and 2±0.2 in BAX (n=3, p<0.05, t-test). In addition, a 3.5±0.5 fold-change reduction in the anti-apoptotic gene BCL-2 was seen (n=3, p<0.05, t-test). No changes in apoptotic gene expression were seen in either sorafenib or imatinib treated cells. Further examination of apoptotic mechanisms using TUNEL staining and immunocytochemistry identified a late apoptotic phenotype in cells treated with sunitinib (2 µM) for 72 hours. These data demonstrate that all three drugs are toxic to human endogenous cardiac stem cells and reduce cell viability. We have further shown that exposure to sunitinib caused cardiac stem cell apoptosis, whereas sorafenib and imatinib appear to be linked to necrosis.