Our goal is to generate a toolbox of human embryonic stem cell lines to monitor blood forming (hematopoietic) stem cell development in culture. Currently, we lack markers that we can use to pinpoint the developmental stage of human ES cell derived candidate hematopoietic stem cells (HSC), and whether they have established the correct molecular machinery required for proper function. Our studies have revealed that correct pattern of HOXA gene activity is essential for specifying human HSCs and maintaining their function. We have generated fluorescent reporter hESC lines for HOXA9 and HOXA10 genes, and are preparing the constructs to target the earlier HOXA genes. In addition, we have generated hESC lines that express a fluorescent reporter from the regulatory elements of novel HSC genes, MLLT3 and HLF. Our next goal is to determine culture conditions that will generate HSCs that are able to turn on and maintain the expression of these important HSC regulators. This knowledge will be critical for generating HSCs in culture dish for transplantation therapies to treat leukemias and other life-threatening blood disorders.