(See Supplemental Table S3 for a list of genes with differentially occupied EBF1 peaks.) ((((in both Dox-treated EBF1wt- and EBF1E271A-expressing pro-B cells (Supplemental Fig. cell specification. The transcriptional network in which EBF1 operates has been extensively studied; however, the regulation of EBF1 function remains poorly defined. By mass spectrometric analysis of proteins associated with endogenous EBF1 in pro-B cells, we identified the nuclear import receptor Transportin-3 (Tnpo3) and found that it interacts with the immunoglobulin-like fold domain of EBF1. We delineated glutamic acid 271 of EBF1 as a critical residue for the association with Tnpo3. EBF1E271A showed normal nuclear localization; however, it had an impaired B cell programming ability in conditions of Notch signaling, as determined by retroviral transduction of in mice resulted in a block of early B cell differentiation, accompanied by a down-regulation of B lineage genes and up-regulation of T and NK lineage genes. Taken together, our observations suggest that Tnpo3 ensures B cell programming by EBF1 in nonpermissive conditions. Keywords: EBF1, Notch1, B cell programming, B lymphopoiesis The commitment of hematopoietic progenitors to the B cell lineage requires the activity of lineage-specific transcription factors that induce a B lineage-specific gene expression Lanatoside C program and/or repress genes associated with alternative cell fates (for reviews, see Nutt and Kee 2007; Boller and Grosschedl 2014; Rothenberg 2014). Multipotent progenitors differentiate in the fetal liver and in the bone marrow via common lymphoid progenitors, pre-pro-B cells, pro-B cells, and pre-B cells to immature B cells that display an antigen-specific receptor on their cell surface. This developmental process includes multilineage priming of enhancers, which provides permissive chromatin states for the action of lineage-specific transcription factors (Heinz et al. 2010; Mercer et al. 2011; Strid et al. 2021). Moreover, a regulatory networkconsisting of the transcription factors E2A, Foxo1, EBF1, and Pax5has been implicated in the specification of the B cell lineage and the activation of B cell genes (Medina et al. 2004; Lin et al. 2010; Mansson et al. 2012). In this network, EBF1 has a lineage-instructive function because it is sufficient to overcome an early differentiation block of progenitors lacking the transcription factor Ikaros (Reynaud et al. 2008). EBF1 Lanatoside C acts as a pioneer transcription factor that binds na?ve, inaccessible chromatin and mediates a de novo gain of chromatin accessibility by recruiting chromatin remodelers (Maier et al. 2004; Boller et al. 2016; Li et al. 2018; Wang et al. 2020; Strid et al. 2021). In addition, Pax5 and EBF1 are also involved in committing cells to the B cell fate by repressing genes associated with alternative lineage potential (Nutt et al. 1999; Cobaleda et al. 2007; Nechanitzky et al. 2013). In particular, Pax5 represses genes encoding cell surface receptors, such as Notch1 and CSF1R, whereas EBF1 represses RSTS genes coding for T lineage-promoting transcription factors, such as TCF1 and GATA3 (Souabni et al. 2002; Revilla et al. 2012; Banerjee et al. 2013; Nechanitzky et al. 2013). Notch1 is a key determinant for specifying the T lineage that is activated in early thymic progenitors (ETPs) by the interaction with ligands of Lanatoside C Delta-like family on stromal cells of the thymus (for reviews, see Maillard et al. 2005; Tanigaki and Honjo 2007; Radtke et al. 2013). This interaction leads to the cleavage of the Notch intracellular domain (NICD), which interacts with the transcription factor RBPJ on Notch-responsive genes (Jarriault et al. 1995). NICD activates the genes encoding the T lineage transcription factors TCF1 and GATA3, which activate T cell-specific gene expression in combination with other transcription factors (Hozumi et al. 2008; Germar et al. 2011). Notch signaling in ETPs results in the repression of and (Zhang et al. 2012), and Notch1 has been implicated in antagonizing the function of EBF1 in transient transfection assays (Smith et al. 2005). Thus, the cross-antagonistic features of EBF1 and Notch signaling may govern the early cell fate decisions.