Cup-like nuclear invaginations (NIs) in acute myeloid leukemia (AML) blasts have been associated with NPM1 mutations. Precision for enumeration of NI blasts has not been previously studied. Furthermore, the sensitivity and specificity for the morphologic prediction of NPM1 mutations have been variously reported. By using 66 AML specimens (17 with NPM1 mutations and 49 without), we found that interobserver reproducibility for enumeration of NI blasts was high (r = 0.98) and that identification of this feature was teachable (r = 0.96). No NPM1 mutation-negative case had greater than 7% NI blasts. The fraction of NI blasts was highly variable among 17 NPM1 mutation-positive cases, ranging from 0% to greater than 40%. These data indicate that an NI blast fraction of more than 10% is highly specific for NPM1 mutation-positive cases but with a sensitivity of about 30%. Therefore, although NI blasts can be reliably identified in routine smears and although they are a specific marker of NPM1 mutation-positive cases, the majority of NPM1 mutation-positive cases lack this distinctive finding.