Despite recent progress in identifying the genetic drivers of acute lymphoblastic leukemia (ALL), prognosis remains poor for those individuals who experience disease recurrence. Moreover, acute leukemias of ambiguous lineage lack a biologically informed framework to guide classification and therapy. These needs have driven the adoption of multiple complementary single-cell sequencing approaches to explore key issues in the biology of these leukemias, including cell of origin, developmental hierarchy and ontogeny, and the molecular heterogeneity driving pathogenesis, progression, and therapeutic responsiveness. There are multiple single-cell techniques for profiling a specific modality, including RNA, DNA, chromatin accessibility and methylation; and an expanding range of approaches for simultaneous analysis of multiple modalities. Single-cell sequencing approaches have also enabled characterization of cell-intrinsic and -extrinsic features of ALL biology. In this review we describe these approaches and highlight the extensive heterogeneity that underpins ALL gene expression, cellular differentiation, and clonal architecture throughout disease pathogenesis and treatment resistance. In addition, we discuss the importance of the dynamic interactions that occur between leukemia cells and the nonleukemia microenvironment. We discuss potential opportunities and limitations of single-cell sequencing for the study of ALL biology and treatment responsiveness.