The application of Systems Biology methods in the emerging field of Glycomics requires the collection and integration of glycosyltransferase data at the gene and enzyme level for the purpose of model building. In this study, we systematically examined the relationship between gene expression, glycosyltransferase activity and selectin-binding function in: i) human neutrophils, ii) undifferentiated HL-60 (human promyelocytic leukemia cells), iii) HL-60 differentiated along granulocytic lineage. Emphasis was placed on glycosyltransferase enzymes mediating the formation of sialyl Lewis-X (sLeX) type carbohydrate structures, since these structures recognize adhesion molecules belonging to the selectin family. Also, such carbohydrate structures on a leukocyte glycoprotein, P-selectin Glycoprotein Ligand-1 (PSGL-1), was the focus of this study since this is an important selectin ligand. Real time PCR was used to monitor glycosyltransferase transcript levels, and synthetic carbohydrate acceptors were used for measuring enzymatic activity of individual glycosyltransferases. Our results suggest that FT-VII and ST3Gal-VI likely contribute to the formation of sLeX epitope on O-glycans in neutrophils, while FT-IV/-VII and ST3Gal-IV are the dominant enzymes in HL-60. Thus, distinct biochemical pathways may contribute to the expression of sLeX in human neutrophils and HL-60. The estimates of reaction rate constants under in vitro assay conditions indicate that the enzyme activities contributing to sLeX formation in leukocytes vary as: ST3[Galβ1,4GlcNAc] < β1,3GlcNAcT £ α1,3FT. HL-60 differentiation was accompanied by changes in the mRNA levels of PSGL-1 and various glycosyltransferases that contribute to sLeX epitope. As a result, the alterations in glycan structures led to reduced P-selectin binding to cell surface PSGL-1 upon cellular differentiation. Overall, our study suggests that glycosyltransferase gene expression and enzyme activity measurements can be combined with existing knowledge of glycobiology to semi-quantitatively predict glycan structures, identify key rate controlling steps in carbohydrate ligand formation and generate experimentally testable hypothesis.