However, exactly what stages are most permissive for transformation remains imprecisely defined. Furthermore, perturbations during normal B-cell differentiation can lead to development of B-cell acute lymphoblastic leukemia (B-ALL) 11, 12, 13. Capturing transition states as B cells differentiate from one stage to the next is particularly difficult. These limitations have led to an incomplete understanding of B-lymphoid transcription-factor expression kinetics across the B-cell developmental trajectory, and the orchestration of transcriptional programs underlying the alternating cycles of proliferation and differentiation. However, these markers are insufficient to fully demarcate distinct subsets 1, resulting in the analysis of mixed populations. The use of such markers in combination with distinct gene knockout mice has greatly expanded our understanding of specific B-lymphoid transcription factors 5, 6, 7, cytokines 8, 9, 10, and signaling pathways that entrain B cell development. Our findings demonstrate the underlying heterogeneity of developing B cells and characterise developmental nodes linked to B cell transformation.ĭistinct stages of B-cell development have been delineated using flow cytometry and a variety of surface 1, 2 and intracellular markers 3, 4. Using pseudotime analysis, we further characterize the expression kinetics of different biological modalities across B cell development, including transcription factors, cytokines, chemokines, and their associated receptors. These changes correlate with reciprocal changes in expression of the transcription factor EBF1 and the RNA binding protein YBX3, that are defining features of the pre-BCR-dependent stage. Here we show unique transcriptional signatures that refine the pre-B cell expansion stages into pre-BCR-dependent and pre-BCR-independent proliferative phases. We use single-cell transcriptomics/proteomics to identify differentially expressed gene networks across B cell development and correlate these networks with subtypes of B cell leukemia. Integration of external signals and B-lymphoid transcription factor activities organise B cell lineage commitment through alternating cycles of proliferation and differentiation, producing a diverse repertoire of mature B cells.