A hallmark of subclinical atherosclerosis is the build-up of vascular smooth muscle cell (SMC)- like cells and non-SMC cells, particularly macrophage-like cells (MLCs), which cause intimal medial thickening (IMT), lipid buildup, plaque development and blood flow restriction that can lead to a heart attack or stroke. Previous cell fate mapping studies have implicated a putative role for SMCs and resident S100β vascular stem cells (mVSCs) in promoting lesion formation, prior to plaque development. The specific aim of this study was to assess whether murine and human S100β vascular stem cells are capable of myeloid differentiation to MLCs when exposed to atherogenic stimuli cholesterol, oxidized LDL (ox-LDL) in vitro before their response was compared to murine and human SMCs. MLC phenotype was determined by morphological changes, immunocytochemical and immunophenotyping of macrophage surface markers using flow cytometry, in addition to histochemical analysis of lipid droplets and real-time PCR analysis of macrophage phenotypic markers. The data reveal that murine and human SMC-like cells exposed to atherogenic stimuli in vitro increased their expression of myeloid phenotypic markers, typical of MLCs. Moreover, S100β vascular stem cells were also capable of myeloid differentiation but to a greater extent than SMCs in culture in response to the same atherogenic stimuli in vitro. These data suggest that S100β vascular stem cells may represent a novel target for therapeutic intervention to reduce the number of MLCs in atherosclerotic lesions.