B7H3 is a member of the B7 family and is known as an immune checkpoint molecule. B7H3 is known to inhibit T cell function by being expressed in immune cells such as APCs and macrophages. Recent studies have shown that B7H3 is expressed in various cancer cells which affects tumor growth and EMT (epithelial–mesenchymal transition) as well as immune evasion of cancer cells. In addition, our previous study showed that the expression of B7H3 was elevated in cancer stem cell (CSC) derived from MDA-MB453 breast cancer cells compared with non-cancer stem cell (NCSC) through in-situ cell surface biotinylation. This suggests that B7H3 expressed in CSC plays a major role in cancer stem cell immunity, progression and therapeutic resistance of CSC. We have investigated the mechanism by which the expression of B7H3 is increased in CSC and found that transcription of B7H3 was elevated in CSC. Here we performed a DNA pull-down mass spectrometry (DP-MS) with a biotinylated promoter region of B7H3 with CSC and NCSC cell lysates and identified specific transcription factors regulating B7H3 expression in CSC. In this study, we present novel transcription factors that bind to the promoter of B7H3 in CSC using DP-MS method.