Summary: Hematopoietic stem cells (HSCs) comprise a heterogeneous population exhibiting self-renewal and differentiation capabilities; however, the mechanisms involved in maintaining this heterogeneity remain unclear. Here, we show that SATB1 is involved in regulating HSC heterogeneity. Results in conditional Satb1-knockout mice revealed that SATB1 was important for the self-renewal and lymphopoiesis of adult HSCs. Additionally, HSCs from Satb1/Tomato-knockin reporter mice were classified based on SATB1/Tomato intensity, with transplantation experiments revealing stronger differentiation toward the lymphocytic lineage along with high SATB1 levels, whereas SATB1− HSCs followed the myeloid lineage in agreement with genome-wide transcription and cell culture studies. Importantly, SATB1− and SATB1+ HSC populations were interconvertible upon transplantation, with SATB1+ HSCs showing higher reconstituting and lymphopoietic potentials in primary recipients relative to SATB1− HSCs, whereas both HSCs exhibited equally efficient reconstituted lympho-hematopoiesis in secondary recipients. These results suggest that SATB1 levels regulate the maintenance of HSC multipotency, with variations contributing to HSC heterogeneity. : Doi et al. show that hematopoietic stem cells (HSCs) with robust lymphopoietic and long-term reconstituting capability express special AT-rich sequence-binding protein 1 (SATB1). SATB1-expressing and non-expressing HSCs are interconvertible. Moreover, they provide insights into the heterogeneity of HSCs, which are correlated with changes in SATB1 expression levels. Keywords: hematopoietic stem cells, SATB1, lineage bias, heterogeneity, genetic fluctuation, self-renewal, multipotency, lymphopoiesis