Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and remains a leading cause of cancer death in children. Large scale studies examining the genomic landscape of ALL using bulk tumor samples have defined multiple new subtypes, genomic drivers, risk classifying genes and therapeutic targets. However, there are few studies of ALL using single-cell RNA-seq technology to study heterogeneity and the surrounding tumor microenvironment (TME). Previous studies, such as those described here, indicate that single-cell RNA seq studies of AML can provide new insights in the tumor intrinsic and extrinsic factors driving tumor behavior and relapse. Gene expression profiling (GEP) using the 3’ 10x platform of small numbers of matched diagnosis and relapse samples have shown enrichment of a CSF1R signature in the TME at relapse. Single-cell profiling of ALL-stromal cocultures identified a resistant ALL cell population undergoing epithelial mesenchymal transition. Mutational profiling of stem and progenitor populations from leukemia samples was shown to map tumor initiating lesions to developmental stage, indicating that mutational driver and cell of origin is a key determinant of leukemia lineage, and T cell profiling identified autoreactive T cells directed to fusion oncoprotein and mutational neoepitopes. Here, we expand upon previous single-cell studies of ALL, using single-cell RNA seq to profile gene expression, mutational diversity, immunophenotype, TME composition and T cell repertoire in ALL subtypes representative of standard and high risk disease in 95 patients: ETV6-RUNX1-like, KMT2A-rearranged, Ph+, Ph-like, ZNF384-rearranged, B/myeloid mixed phenotype acute leukemia, DUX4-rearranged, MEF2D-rearranged, TCF3::PBX1, hyperdiploid, low hyplodiploid and near haploid ALL. All samples are subject to 5’ 10x single-cell GEP of the tumor, TME and T cell compartments, and B-cell ALL. Simultaneous single-cell cell surface protein sequencing and RNA-seq is incorporated for a subset of tumors with lineage ambiguity. When available, single-cell GEP of relapse samples was obtained. Complementary studies include profiling full length RNA-seq (SMART-seq HT and PacBio) of blast and progenitor cell populations to integrate fusion/mutational profile, expression, and cell of origin.