1 G). process through which the most primitive cells of the hematopoietic system, i.e., hematopoietic stem cells (HSCs), differentiate into mature cells of the myeloid-erythroid and lymphoid lineages (Morrison et al., 1995; Orford and Scadden, 2008; Orkin and Zon, 2008). HSCs are rare and reside in specialized niches located within the BM. In the absence of specific mitogenic stimuli, HSCs remain in a quiescent or dormant state (Trumpp et al., 2010). However, under stress conditions, such as bleeding, myeloablation, total body irradiation, and contamination, HSCs can enter into an active proliferative state (Passegu et al., 2005). Although HSCs are controlled mainly by intrinsic pathways, often HSCs need and respond to external stimuli such as cytokines, chemokines, and cellCcell contacts. Whenever HSCs divide into daughter cells, the fates of the daughter cells, including life versus death and self-renewal versus differentiation, need to be tightly regulated because defects in these cell fate decisions will have detrimental consequences, including BM failure and hematologic malignancies (Passegu et al., 2005). Self-renewal and differentiation of HSCs are complex processes and are dependent on the immediate turning on or turning off of various cytokine receptors, signal transducers, transcription factors, and cell cycle inhibitors. Although transcriptional regulation Calcifediol of gene expression and the involvement of transcription factors in hematopoiesis have been studied to a greater extent, the role of posttranslational modifications (PTMs) of proteins, in particular ubiquitination, in the regulation of hematopoiesis PPARGC1 remains largely unknown. Recent studies, including our own, have highlighted the importance of the ubiquitin proteasome system in the development and functions of normal HSCs and leukemic stem cells (Rathinam et al., 2008, 2010, 2011; Rathinam and Flavell, 2010; Moran-Crusio et al., 2012). Even though these studies have provided clues regarding the physiological relevance of PTMs in hematopoiesis, a clearer understanding of the significance of PTMs mediated by the ubiquitin proteasome system in early hematopoiesis remains elusive. Moreover, these studies were based on the functions of E3 ubiquitin ligases, such as c-Cbl, Itch, and Fbxw7, and the role of deubiquitinases (DUBs) in early hematopoiesis and in stem cell biology needs to be explored. A20 (also known as Tnfaip3 and referred as A20 henceforth) is usually a potent antiinflammatory signaling molecule that restricts multiple intracellular signaling cascades (Ma and Malynn, 2012). A20 is an 90-kD protein that belongs to the ovarian tumor (OTU) family of DUBs. A unique feature of A20 is usually that it contains an N-terminal cysteine protease/DUB domain name (which is necessary for the deubiquitylating functions) and a C-terminal zinc finger (ZNF) domain name (which confers the E3 ubiquitin ligase functions). Thus, A20 can be classified as Calcifediol a dual-function ubiquitin-editing enzyme (Wertz et al., 2004). A20 catalyzes the K48-linked ubiquitylation of target proteins through its C-terminal ZNF domain name, an action which directs the target proteins for proteasomal degradation. Concurrently, A20 removes K63-linked ubiquitin chains from its target proteins (through its DUB activity), which not only inactivates the signaling function Calcifediol of the targets, but might also facilitate its K48-linked Calcifediol ubiquitylation and degradation (Wertz et al., 2004). The unfavorable signaling function of A20 involves deconjugation of K63-linked ubiquitin chains from TRAF6 and RIP1, which are central players of the TLR and TNF Calcifediol receptor (TNFR) pathways (Sun, 2008). In addition, A20 also mediates deubiquitylation of RIP2 and therefore acts as a negative regulator of NF-B signaling (Hitotsumatsu et al., 2008; Sun, 2008; Vereecke et al., 2009; Hymowitz and Wertz, 2010). In view of the fact that A20 has a key role in the control of inflammation and that inflammatory signals can impact HSC development and functions, we hypothesized that A20 functions as a critical regulator of the HSC pool. To validate this, we generated and investigated mice.