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Understanding the regulatory pathways that control the establishment of the different hematopoietic programs and developing methods to identify and isolate progenitors for each are essential to accurately model hematopoiesis in ESC and induced PSC differentiation cultures and ultimately to generate HSCs in vitro. Building on our previous observations that SOX17 is a critical regulator of definitive hematopoiesis (Clarke et al., 2013), in this study, we proceeded to analyze the SOX17-dependent proteome in HECs and hematopoietic progenitors, and through this approach, we were able to identify potential regulatory pathways of the definitive hematopoietic program and markers to distinguish it from the earlier primitive and EMP programs. Specifically, we identified Stat1 as a marker whose eletriptan distinguishes the definitive erythroid lineage from the primitive and EMP-derived erythroid lineages. The differences observed between the EMP and definitive programs are particularly important, as they are difficult to distinguish by other methods. The observation that the EMP program persists through the later definitive stage of differentiation in the Sox17−/− cultures demonstrates that EMP progeny can persist in the PSC-derived differentiation cultures for extended periods of time. Based on these differential patterns of Stat1 expression, we were able to demonstrate that SOX17 dependence is a distinguishing factor that separates the definitive hematopoietic program from the EMP and primitive hematopoietic programs. The expression analyses of the mESC-derived populations showing that the D9 progenitors express Stat1 in a SOX17-dependent fashion provide additional evidence that we were able to specify the definitive program in the differentiation cultures. The analyses of erythroid Stat1 expression will complement the use of T cell potential as a method to distinguish the PSC-derived hematopoietic programs and monitor the emergence of the definitive lineage in vitro. In addition to demonstrating the emergence of the definitive lineage, our studies show that the early specification of the primitive and EMP lineages can be monitored by the co-expression of Stat and globin genes. The observation that the D3.5 FLK-1+ progenitors give rise to both the primitive and EMP erythroid lineages that are found in the yolk sac provides further support that the in vitro PSC system accurately recapitulates in vivo embryonic hematopoiesis. Our STAT protein expression data support the recently published computational prediction of a differential role for STAT signaling in primitive and definitive erythropoiesis (Greenfest-Allen et al., 2013). STAT signaling is central to hematopoietic cell biology, as this pathway is activated in response to numerous cytokines, including erythropoietin (EPO) and stem cell factor (SCF/KL). The observations that EPO activates Stat1, Stat3, Stat5a, and Stat5b in mouse and human cell lines in culture, and that STAT1- and STAT5-deficient fetuses display a wide array of erythroid defects, provide clear evidence that STAT proteins are critical regulators of developmental erythropoiesis and erythroblast maturation (Cui et al., 2004; Halupa et al., 2005; Kirito and Komatsu, 2002; Kirito et al., 1997; Richmond et al., 2005; Socolovsky et al., 1999, 2001). Analysis of the role of STAT3 during erythropoiesis is hampered by the fact that Stat3−/− mice are embryonic lethal at E7.5 due to a lack of mesoderm formation. However, small-molecule inhibition of STAT3 activation resulted in decreased numbers of primitive, but not adult definitive, erythroblasts in culture, suggesting a differential role of STAT3 during distinct stages of hematopoiesis (Greenfest-Allen et al., 2013). A mechanism by which STAT1 may preferentially regulate the maturation of definitive erythroid cells is through its transcriptional target ISG15. ISG15 is strongly upregulated during erythroid differentiation in the bone marrow (Maragno et al., 2011) and was identified in our proteomics screen as a potential SOX17 target exhibiting a parallel expression pattern to STAT1 (Figure 2D). ISG15 can be covalently linked to lysine residues of target proteins as a method of post-translational modification (Zhang and Zhang, 2011). STAT5 and globin proteins have been shown to be ISGylated in erythroblasts, and ISG15−/− erythroblasts are unable to terminally differentiate, thus providing a testable hypothesis by which STAT1 regulates definitive erythroid differentiation. Collectively, the observation that distinct developmental subsets of erythroid cells express different STAT proteins presents a previously unidentified mode of regulation of embryonic erythropoiesis.