Numerous studies have analyzed the expression and prognostic importance of various proteins in acute myelogenous leukemia (AML). We sought to determine whether the sample source and methodology used to measure protein expression affect the results obtained. To determine the importance of sample source, we used Western blotting to compare the expression of eight proteins and phosphoproteins in the leukemia blast-enriched fraction of 118 blood- and 108 marrow-derived samples, including 37 paired samples. To determine the importance of methodology, the expression of five proteins was measured in 20 paired samples by Western blotting, laser scanning cytometry (LSC), and flow cytometry. The mean expression and range of expression in blood- and marrow-derived samples were statistically identical for all eight proteins. Expression measurements for the 37 paired blood and marrow samples also had very high statistical correlation. The LSC and flow cytometry data had the highest concordance when compared using Kolmogorov-Smirnoff D-stats (range of R values, 0.8-1.0). High concordance was also observed between the LSC and flow cytometry results when the percentage of cells positive for expression was dichotomized into positive or negative expression. However, there was less correlation between LSC and flow cytometry when the actual percentages of positive cells were compared. The majority of discordant situations involved samples that were positive by flow cytometry but negative by LSC. The correlation between Western blotting signal intensity and the percentage of expression-positive cells measured by LSC or flow cytometry varied by protein but was limited when there was little heterogeneity in expression by either method. In conclusion, provided that leukemia blast-enriched fractions were analyzed, the blood- and marrow-derived samples had identical protein expression. There was good concordance of results between flow cytometry and LSC, which share similar technology, but more limited correlation between these methods and Western blotting.