Acute leukemia of ambiguous lineage (ALAL) includes mixed-phenotype acute leukemia (MPAL), which exhibits immunophenotypic evidence of differentiation along more than one cell lineage, and acute undifferentiated leukemia (AUL), which lacks sufficient immunophenotypic differentiation along any cell lineage. This review provides an overview of ALAL, emphasizing the central role of flow cytometric analysis in its diagnostic workflow. It primarily focuses on MPAL, addressing updated classification and diagnostic criteria by the WHO-HEM5 and the ICC, including both genetically defined and phenotypically defined MPAL. The article provides a detailed review of the MPAL lineage assignment criteria with an illustrative description of a series of MPAL cases. Future studies are needed to reconcile the different criteria used in these two classifications. Continuously expanded molecular studies are expected to provide a genomic and lineage-associated framework for the classification of ALAL with clinical relevance in the diagnosis and therapy selection.