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 cancer cell biology. hESCs are commonly grown on irradiated mouse embryonic fibroblasts (MEFs) or in conditioned medium from MEFs. These culture conditions interfere with many experimental conclusions and limit the ability to perform conclusive proteomics studies. The current investigation avoided the use of MEFs or MEF-conditioned medium for hESC culture, allowing global proteomics analysis without these confounding conditions, and elucidated neural cell-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 proteins 2 and 4 and the nuclear autoantigenic sperm protein 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. Analysis of global proteomics, quantitative RT-PCR, and MRM data led to testing the isoform interference hypothesis where redundant peptides dilute quantification measurements of homologous proteins. These results show that targeted MRM analysis on nonredundant peptides provides more exact quantification of homologous proteins. This study 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. Molecular & Cellular Proteomics 7:750 -767, 2008.Human embryonic stem cells (hESCs) 1 are perhaps the most promising source of cells for regenerative medicine and treatment of disease. Despite extensive research aimed at elucidating and controlling the processes of self-renewal and lineage-specific differentiation, much remains to be learned regarding the basic cell biology of hESCs before their clinical potential can be realized. Our current knowledge regarding the complex regulation of lineage segregation during development arises primarily from in vivo investigations in invertebrate and mouse. Furthermore manipulation of the signaling pathways initiating and controlling lineage differentiation during development, including the role of bone morphogenic proteins (BMPs), has been expedited by in vitro studies of mouse embryonic stem cells (mESCs). Only recently has considerable research commenced on hESCs.BMPs were originally characterized based on their ability to induce cartilage and bone formation. They are now known to be multifunctional regulators of development, including many non-osteogenic processes (1). There are as many as 30 different BMP family members classified according to...