SUMMARY We discovered that MLLT3 is a key regulator of human hematopoietic stem cell (HSC) self-renewal but its expression declines in culture. Strikingly, maintaining MLLT3 levels in cultured cord blood (CB) HSCs results in expansion of transplantable HSCs without transformation or differentiation bias. How MLLT3 dictates physiological HSC self-renewal without transforming them to leukemic state, became a major question. We discovered that MLLT3 locus in human HSPCs has a second active TSS linked to hitherto unreported short MLLT3 isoform (MLLT3-S). MLLT3-S encodes a truncated protein that retains the AHD domain responsible for protein-protein interactions but lacks the chromatin binding YEATS domain. Overexpressed MLLT3-S can interact with known MLLT3 protein partners such as DOT1L and superelongation complex but cannot bind chromatin. While MLLT3-L OE in CB HSCs promoted their expansion, MLLT3-S OE suppressed it, implying distinct functions for the two isoforms in HSCs. Conversely, knockdown (KD) of MLLT3-L in CB HSCs triggered premature differentiation, whereas MLLT3-S KD resulted in relative expansion of immunophenotypic HSCs, although they engrafted poorly in immunodeficient mice, showing that both isoforms of MLLT3 are necessary for proper HSC function. scRNA and bulk RNA seq of HSPCs with MLLT3-L and/or MLLT3-S OE and KD revealed that the two isoforms have opposing effects on expression of key HSC processes, such as mitochondrial biosynthesis and oxidative phosphorylation, translation, as well as several HSC transcriptional regulators. We discovered that, while MLLT3-L is expressed already in hemogenic endothelium in the embryo, MLLT3-S is induced in maturing fetal liver HSCs and represses fetal programs in HSCs, implying that MLLT3-S may promote HSC maturation in the fetal liver and their transition to homeostatic state. We have identified several candidate enhancers in MLLT3 gene that link to either the long or short isoform. Interestingly, building on our discovery that there are two major isoforms of MLLT3 that are transcribed from the two TSS, correlation of our various sequencing data with current Gencode and Ensembl annotations suggests that both MLLT3-L and MLLT3-S may have less frequent subvariants. These potential sub-variants are likely generated by alternative splicing and may lead to dysfunctional proteins or non-translated transcripts, suggesting potential complementary mechanisms by which the function of MLLT3 isoforms can be dampened or modulated. We will now examine the mechanisms by which the interplay between long and short isoforms of MLLT3 in human HSCs regulate HSC maturation and the balance between expansion and maintenance modes by 1) mapping the expression of MLLT3-L and S (and their prevalent sub-variants, if present), and their associated regulatory elements throughout human HSC ontogeny, by 2) determining functional effects of MLLT3 long and short isoforms (and their sub-variants) on human HSC maturation and fa...