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 (ALAL) 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 molecular heterogeneity driving pathogenesis, progression, and therapeutic responsiveness in ALL and related leukemias. There are multiple single cell techniques to profile a specific modality, including RNA, DNA, chromatin accessibility and methylation, and an expanding range of approaches to simultaneously analyze more than one modality. 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 dynamic interactions occurring between leukemia cells and the non-leukemia microenvironment. We discuss potential opportunities and limitations of single-cell sequencing for the study of ALL biology and treatment responsiveness.