Elucidating the complex combinations of growth factors and signaling molecules that maintain pluripotency or, alternatively, promote the controlled differentiation of human embryonic stem cells (hESCs) has important implications for the fundamental understanding of human development, devising cell replacement therapies, and for cancer cell biology. hESCs are commonly grown on irradiated mouse embryonic fibroblasts (MEFs) or in conditioned media from MEFs. These culture conditions interfere with many experimental conclusions and limit the ability to perform conclusive proteomic studies. The current investigation avoided use of MEFs or MEF conditioned media for hESC culture, allowing global proteomic analysis without these confounding conditions and elucidated neural-specific signaling pathways involved in noggin-induced hESC differentiation. Based on these analyses, we propose the following early markers of hESC neural differentiation: collapsin response mediator protein (CRMP) 2 and 4, and the nuclear autoantigenic sperm protein (NASP) as a marker of pluripotent hESCs. We then developed a directed mass spectrometry assay using multiple reaction monitoring (MRM) to identify and quantify these markers, and in addition, the epidermal ectoderm marker cytokeratin-8, and macrophage migration inhibitory factor (MMIF). Analysis of global proteomics, qRT-PCR and MRM data lead to testing the isoform interference hypothesis where redundant peptides dilute quantification measurements of homologous proteins. These results show, targeted MRM analysis on non-redundant peptides provides more exact quantitation of homologous proteins. This manuscript describes the facile transition from discovery proteomics to targeted MRM analysis and allowed us to identify and verify several potential biomarkers for hESCs during noggin-induced neural and BMP4-induced epidermal ectoderm differentiation.